Alkaline microbial enhanced oil recovery

ABSTRACT

A method of enhanced oil recovery that includes water-flooding an oil reservoir with an alkaline fluid or a fluid containing a compound toxic to indigenous microbes and inoculating the oil reservoir with a consortium comprising microbes that naturally are, or are engineered to be, obligatory alkaphilic, halo-alkaphilic or alkaline tolerant, and naturally are, or are engineered to be, deficient in their ability to utilize short chain hydrocarbons of 12 carbons or less but have the ability to convert hydrocarbons into fatty acids.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 13/385,520filed Feb. 23, 2012, which claims priority under Section 119(e) and thebenefit of U.S. Provisional Application Nos. 61/446,674 filed Feb. 25,2011, 61/496,461 filed Jun. 13, 2011 and 61/633,785 filed Feb. 17, 2012,the entire disclosures of which are hereby incorporated by reference intheir entirety.

FIELD OF THE INVENTION

The present invention is from the field of microbial enhanced oilrecovery (MEOR). In particular, the invention concerns new, efficient,economical and environmentally safe microbial methods to enhance oilrecovery, as well as microorganisms useful in such methods.

BACKGROUND OF THE INVENTION

The demand for crude oil has exceeded the existing production in theUnited States for more than 30 years, which has led to increasing demandfor more imported oil and a dependency on foreign suppliers. Any newtechnology that could increase the efficiency of oil recovery would beof great benefit to countries such as the U.S. that have large amountsof unrecoverable oil in place (OIP) in older exiting oil fields.

Most of the remaining undeveloped oil in the Western Hemisphere is notlight petroleum, but is heavy oil or tar sands. Large deposits of heavyoil are in Venezuela and California. Canada has large deposits of tarsands. Currently, production of heavy oil requires large amounts ofenergy.

Most petroleum is found in sandstone, siltstone or carbonate. Unlikenatural gas, the recovery of petroleum oil is not efficient. Theexisting conventional oil production technologies are able to recoveronly about one-half of the oil originally in place in a reservoir oflight oil. For heavy oil, the recovery is often less than 10%. Tar sandsare so heavy that they will not flow at all and no oil can be recoveredby conventional drilling and pumping. A technology that could recover agreater percentage of this residual oil could increase oil productionfrom existing reservoirs and reduce the need of the U.S. for importedoil. The additional oil recovered from existing oil producing reservoirscould reduce the need to explore and develop wilderness areas that arepotential new oil fields. This additional recovery of existing oil couldbridge the gap needed for the development of alternative renewableenergy sources.

The Original Oil In Place (OOIP) is the petroleum present in the oilreservoir when first discovered. The volume of the reservoir isdetermined by the size and porosity of the carbonate or sand stone. Theporosity of the rock is a measure of the amount of small chambers ormicro-traps within the rock that can hold water or oil. The oil isgenerally pushed up to the surface with the existing oil reservoirpressures at first, but the pressure in the oil well drops with time.Therefore, there is a need to create overpressure with other means suchas water injection or a gas injection for secondary recovery of theOOIP. The choice of a specific secondary recovery technique depends onthe type of the hydrocarbon accumulation and the nature of thereservoir. Water injection or “water sweep” or “waterflooding” is acommon secondary recovery technique. In waterflooding, pressurized wateris injected into the oil-bearing formation rock. Ideally, the injectedwater displaces the residual oil and moves it to a producing well.Generally in waterflooding, crude oil free of water is recovered first,and then subsequently a mixture of crude oil and water are recoveredfrom the production wells. At some point, the percentage of water in theoil-water mixture (referred to as the water cut) from this techniquebecomes so high that it is uneconomical to continue pumping oil from thewell. The problem, with using water as a “drive fluid”, is that waterand oil are immiscible. The lower viscosity water will flow over the oiland by-pass large amounts of oil. Therefore, even after secondaryrecovery, a significant portion of crude oil remains in the formation,in some cases up to 75% of the OOIP. The fraction of unrecoverable crudeoil is typically highest for heavy oils, tar, and large complexhydrocarbons. In the U.S. this residual OIP in old oil wells could be asmuch as 300 billion barrels of light oil. World-wide, the estimate ofunrecoverable oil is 2 trillion barrels. There are an additional 5trillion barrels of heavy oil, most of which is unrecoverable. Much ofthis remaining oil is in micro-traps due to capillary forces or adsorbedonto mineral surfaces (irreducible oil saturation) as well as bypassedoil within the rock formation.

Enhanced Oil Recovery

Oil recovery by injection of fluids not normally found in the reservoiris referred to as Enhanced Oil Recovery (EOR). It is a subset ofImproved Oil Recovery (IOR), which can include operational strategiessuch as infill drilling and horizontal drilling. Although it issometimes referred to as tertiary recovery, it can be implemented alongwith secondary processes. Many types of EOR have been proposed and usedover the years. Technical complexity and the high cost of chemicals haveprevented the widespread use of EOR to where it only represents about10% of total United States oil production.

There have been two major EOR approaches; thermal and non-thermal.

Thermal Processes

Thermal processes work by heating the reservoir rock and the oil toreduce viscosity of the heavy oil. In general, the lower the viscosityof the oil, the better its recovery will be. The most widely usedthermal process is steam injection in which the temperature of thereservoir and the remaining oil is increased by heat energy of steam.Hot water may also be used, but it is not as efficient at transferringheat to the oil and rock in the reservoir. Unfortunately, in bothprocesses, most of the heat energy is lost to the surroundings and doesnot go to heating the oil. In situ combustion of the oil is much moreefficient than steam because it only heats the reservoir and not all thepipes and overburden rock. However, in situ combustion is difficult tocontrol and is seldom used. Typically, it requires the energy equivalentof a half a barrel of oil to recover a barrel of oil with a steaminjected thermal process. However, this depends on the oil saturationand the configuration of the reservoir. Because most of the energycarried by the steam is given up to the pipes, wall rock, and reservoir,it is best to use only on reservoirs with a high oil content so as torecover as much oil as possible with the steam used to heat thereservoir rock. Generally, thermal methods are used on heavy oil becauseit reduces the viscosity of the oil and increases the mobility of theoil and the mobility ratio (mobility of displacing fluid to mobility ofdisplaced fluid or oil). Typically, recoveries are in the range of 50 to60% for a thermal process, but the net energy gain is much less thanthat because of the large amount of energy needed to make steam. Theideal situation for thermal oil recovery is when there exists a nearbysource of inexpensive or waste energy for steam generation.

Non-Thermal Processes

Non-thermal methods are best suited for light and moderately viscousoils. The major objectives for these processes are to lower theinterfacial tension (IFT) between the oil and displacing fluid and toimprove the mobility ratio. Many of the non-thermal processesexperimented with or used over the years rely on surfactants forreducing the oil viscosity and decreasing the IFT between the oil anddisplacing fluid. Ideally, the mobility of the displacing fluid shouldnot be higher than the oil. The mobility ratio (mobility of displacingfluid over mobility of displaced fluid) should be low. The mobility ofthe oil can be increased by viscosity reduction and by IFT reduction. Asthe IFT is decreased, the oil becomes more miscible with the fluid untilit becomes one phase and the IFT is zero. This decreases the mobilityratio and increases the oil recovery. Alternatively, the viscosity ofthe displacing fluid can be increased by adding polymers to “thicken”the liquid. Non-thermal methods require less energy and are best suitedfor light oil of 100 cp or less. However, most non-thermal methodsrequire considerable laboratory experimentation and processoptimization. The high cost of surfactants and polymers is generally thelimiting factor for chemical EOR.

There are two major classes of chemical or non-biological EOR. One ismiscible flooding with a displacing fluid that is miscible with thereservoir oil and will reduce the IFT to zero. The displacing fluids canbe solvents such as propane or pentane or compressible gases that arealso soluble in the oil. The temperature of the reservoir must be lowenough so that the gas can be compressed to a liquid at the pressurethat the reservoir can withstand without fracturing. Some examples ofcompressible gases are: natural gas, flue gas, nitrogen and carbondioxide. Carbon dioxide has been gaining in prominence in recent years,partly due to the possibility of green house gas sequestration. Theamount of carbon dioxide required to recover oil is substantial(500-1500 sm³/sm³ oil). Although these processes can recover up to 20%of the OOIP, their use is limited to a fraction of all reservoirs due toreservoir pressure and temperature requirements and availability ofgases. Currently, in over 80% of all carbon dioxide gas EOR projects,the gas is delivered to the well site by pipeline from deep carbondioxide mines in a few locations in the US.

The other major class utilizes a chemical formulation as the displacingfluid. The chemical compounds interact with the oil or the water or bothin such a way that there is a decrease in mobility ratio and IFT whichleads to better oil mobility and recovery. Chemical methods have a majoradvantage over both thermal and compressed gases in that they generallyhave lower capital requirements and are not limited by location andavailability of gases or sources of inexpensive heat energy. Economicsis the major deterrent to the use of chemical EOR. Many of the chemicalsused in these processes are manufactured from petroleum and their costincreases as the price of oil increases.

There are four major chemical flooding processes.

Polymer flooding functions by improving the mobility ratio and reducingthe permeability contrast of the reservoir. In most cases a slug ofpolymer solution of about 20 to 40% of the reservoir pore volume ispumped into the injection wells. Losses of polymer to the porousreservoir rock and degradation of the polymer due to shear forces canlimit the success of the method. The polymers can be synthetic chemicalpolymers such as polyacrylamide or biologically produced such aspolysaccharides. Some bio-polymers are more effective at high salinitythan the chemical polymers, but are also more expensive to produce.

Surfactant flooding is effective by lowering the IFT between oil andwater. A surfactant molecule has a polar group on one end of themolecule and a hydrophobic regain on the other end of the molecule. Theideal surfactant is one that will reside in both the oil phase and waterphase at the oil water interface. Petroleum sulfonates or otherpetroleum compounds with a charged or polar group are often used assurfactants. Excessive loss of surfactant to reservoir rock surface andthe high cost of surfactant production have limited the use of thisprocess. However, surfactants can be used in combination with otherchemical EOR methods to increase the performance.

Alkaline flooding and alkaline-surfactant-polymer (ASP) flooding takesadvantage of acid compounds naturally found in some petroleum. Inalkaline flooding, an aqueous solution of alkaline chemicals, such assodium hydroxide, sodium carbonate or sodium bicarbonate is injectedinto a reservoir. The alkaline chemicals react with the acid compounds,also referred to as naphthenic acid, of the crude petroleum oil to formin situ surfactants on the surface of the oil. This causes a reductionin IFT and sometimes a spontaneous emulsification of the oil. Thealkaline flooding is followed by a slug of surfactant and polymers insolution which can significantly increase oil recovery. The alkali alsoreduces adsorption of surfactant onto the surface of the formation rockand thereby decreases cost.

This process is limited to oil that has sufficient organic acid to betransformed into suitable surfactants. The amount of acid in thepetroleum reservoir oil can be determined by extraction with base andthen titration by hyamine or by direct titration of acid in an organicsolvent. This analysis generates an acid number which is defined as themilligrams of potassium hydroxide need to neutralize the acids presentin one gram or oil. It is generally believed that the target oil musthave an acid number of 0.4 or more to be amenable to alkaline flooding.However, this is only approximate because a simple acid number does notprovide details on the type of acids present in the oil. The directtitration of all the acid in oil is called the total acid number (TAN)and is generally much higher than the extracted acid titrated withhyamine. The TAN number is misleading because large hydrocarbon acidsare too lipophilic to be extracted from the oil by dilute sodiumhydroxide solution. These large lipophilic acids will also not functionas good surfactants or soaps at the oil-water interface. Smallhydrocarbon acids are too hydrophilic to be detected by the hyaminetitration and are also not useful as soaps because they move into theaqueous phase and do not help lower the IFT of the oil. Therefore thebest measure of the naphthenic acid is an aqueous extraction andtitration with hyamine.

For petroleum reservoirs that contain unrecovered oil with anextractable acid number of 0.4 or more ASP flooding can be the lowestcost chemical EOR process. Unfortunately, most petroleum reservoirs inthe U.S. do not have a sufficiently high enough extractable acid to beamenable to ASP flooding. Some shallow oilfields have high TAN which isbelieved by some to be the result of many years of microbialdegradation. However, this slow natural process has also removed most ofthe alkanes and other lighter oil compounds leaving the residual oilvery viscous. In 1998 A. K. Stepp and T. French proposed a process offirst biodegrading oil to increase the TAN in order that the oil wouldbe more amenable to ASP or alkaline flooding. The limitation of thisproposed two step process is that the lighter molecular weight alkanesand aromatic hydrocarbons will be converted faster to fatty acids thanthe higher molecular weight hydrocarbons. Another problem is that manyof the fatty acids that are produced will also be utilized as a carbonsource by the injected microbes and the indigenous microbes.

Another limitation of a two step process is the first step ofbiodegrading the oil to increase the acid content can be a long processtaking many months or years. The actual time needed to complete thebiological conversion of hydrocarbons to fatty acids is variable andunpredictable. The ability to determine the actual acid content in theresidual oil trapped within the underground reservoir is limited.Drilling into the reservoir formation for oil analysis is very expensiveand is not a reasonable meriting technique. Starting the alkalineflooding too soon before enough acid were generated would not produceenough oil recovery because the TAN was too low. Delaying the start ofthe alkaline process would cause more degradation of light hydrocarbonsand a loss of the light oil fraction with an increase in viscosity whichwould also delay the start of oil production.

Therefore there is a need to combine microbial oil degradation withalkaline and ASP flooding in such a way that high oil recovery can beachieved without loss of oil to extensive bio-degradation or lengthymultistep processes. The need is for a new alkaline process that it canbe used on a larger number of reservoirs and that would reduce thechemical cost of the surfactants and polymers.

Microbial Enhanced Oil Recovery (MEOR)

One special type of EOR technique uses microorganisms such as bacteriaand Achaea to dislodge the micro-trapped or adsorbed oil from the rock.The goal of this technique, which is known as microbial enhanced oilrecovery (MEOR), is to increase oil recovery of the original subsurfacehydrocarbons using microbes rather than the more costly chemicalrecovery processes. These biological processes typically usemicroorganisms to achieve similar results as the chemical methods inthat they reduce IFT and reduce the mobility ratio of the water drivefluid to oil. The major mechanisms by which microbes are believed tofunction by are: (1) alteration of the permeability of the subterraneanformation by producing low molecular weight acids from thebiodegradation of hydrocarbons that cause rock dissolution, (2)production of biosurfactants that can decrease IFT and form micelles ofoil in water in a way similar to chemical surfactants, (3) mediation ofchanges in wet-ability of the oil droplet by growing on the droplet andchanging the surface of the oil to a less hydrophobic surface (4)production of bio-polymers that improve the mobility ratio of water topetroleum by increasing the viscosity of water and plugging high flowchannels, (5) production of lower molecular weight hydrocarbons byenzymatically converting the large hydrocarbons into smaller molecules,which will reduce of the oil's viscosity, (6) generation of gases(predominantly carbon dioxide and nitrogen) that increase formationpressure.

Of all the EOR processes, MEOR is presently considered the lowest costapproach, but it is generally the least often used. One of thelimitations of MEOR processes that stimulate indigenous microbes is thatthere is little control of the six proposed mechanisms of biological oilrecovery. It is also possible that other unknown mechanisms areresponsible for the more successful field tests of MEOR. Without betterunderstanding or control of this biological process it is unlikely to beused to recover oil from large oil fields. In order to be used as otherchemical or thermal oil production processes, it would be best if eachof the above mechanisms could be tested separately.

Numerous microorganisms have been proposed for achieving variousmechanisms of the microbial mobilization process in subterraneanformations. Field tests of these microbes involved injection of anexogenous microbial population into old and low producing oil wells. Theinoculating culture was supplied with nutrients and mineral salts asadditives to the water pumped into wells for oil recovery. Thedevelopment of exogenous microorganisms has been limited by theconditions that prevail in the formation. Physical constraints, such asthe small and variable formation pore sizes together with the hightemperature, salinity and pressure of fluids in the formation and thelow concentration of oxygen in the formation waters severely limit thetypes and number of microorganisms that can be injected and thrive inthe formation. Later, it became apparent that indigenous microbesstimulated by the nutrients were playing the major role in oil recovery.Accordingly, it is difficult to determine which of the variousbiological mechanisms were at work.

Biological constraints, such as competition from indigenous microbes andthe stress of changing environments (from surface to subsurface) alsoact to limit the viability of exogenous microorganisms. To overcomethese problems, the use of indigenous microorganisms, commonlyanaerobic, has been proposed in MEOR projects. It is known that bacteriaand other microbes can grow indigenously within petroleum oil reservoirsand can be used to enhance oil production. It is also known thatbacteria and other microbes will metabolize various components ofpetroleum as a carbon and energy source. In addition to the beneficialeffects of making surfactants, solvents and other metabolites that canresult in an increase in oil production; they can consume oil as acarbon source. Unfortunately, they generally prefer to consume theshort-length alkanes.

In fact, the process of petroleum bio-degradation relies on theemulsification of oil so that the hydrocarbon can be transported intothe bacterial cells for conversion to fatty acids as a carbon and energysource. This process can be used to remediate oil spills and other oilcontaminated sites by supplying the indigenous microbes with nutrientsor inoculating with cultures of microbes that can degrade oil. In thecase of biological remediation of petroleum contaminated sites, microbescan produce metabolites such as surfactants that help emulsify oil sothat they can then use the emulsified oil as a carbon source. Both ofthese functions help remove the hydrocarbon contamination from the site.However, in the case of MEOR only the production of metabolites such assurfactants, bio-polymers, hydrocarbon cleaving enzymes, organic acidsand solvents are beneficial to increased oil production. Other thanproviding an energy source, the consumption of light petroleum is notbeneficial to enhanced oil production from the reservoir.

The biodegradation of the shorter carbon alkane chains reduces thelighter fraction of the hydrocarbon mixture in the petroleum oil. Theremoval of the short chain alkanes from this mixture increases theoverall viscosity of the hydrocarbon mixture. The higher viscositymixture is more difficult to recover from the reservoir. The percent ofrecoverable oil is decreased. Also, oil that is recovered is moredifficult to transport through pipes and to refine. Therefore theproduction of useful compounds, by microbes for improved oil recovery,comes with a high cost.

This process of stimulating all the indigenous microbes in an oilreservoir by adding nutrients is unpredictable. The growth of themicrobes may produce the beneficial effect of dislodging oil entrappedwithin a petroleum reservoir. Alternatively, the light oil consumptionmay make the oil more viscous and lower the total recovery of oil.

It would be less detrimental if all petroleum components were degradedequally, but the case is that the shorter chain alkanes and lowermolecular weight aromatics are more readily degraded by the microbes ascarbon and energy sources. Therefore, unless genes that code for shortchain alkane or light aromatics are absent in all microorganisms bothinjected and indigenous it is likely that light hydrocarbon degradationwill be faster than heavy hydrocarbon degradation. This is supported bythe fact that petroleum deposits near the surface, and most subject tobiodegradation, are generally rich in high viscosity oil and containhigh levels of asphaltic hydrocarbon and fairly low on light (short)chain alkanes. Canadian tar sands are believed to be the heavy residuerepresenting about 10% of the original petroleum deposit from which 90%of the oil has been degraded.

In the past, others have taught ways of augmenting the growth ofmicrobes that dislodge and mobilize oil from underground petroleumreservoirs. These methods generally recommend adding nutrients. Somehave also taught adding various cultures of selected bacteria that addedbeneficial capabilities. Some have even reported isolating microbes thatcan only degrade higher molecular weight hydrocarbons (see, e.g. U.S.Pat. No. 5,013,654). However, adding these selected cultures is notenough to achieve the desired result. Although these prior methodsdisclosed that microbes do exist that can only feed on high molecularweight oil, they failed to provide methods of increasing thebio-digestion of heavy oils, while suppressing the lighter weighthydrocarbon consumption by other indigenous microbes. The microbes thatare naturally residing within the petroleum reservoir are likely to havethe ability to degrade lower weight oil. Adding nutrients will generallystimulate the growth of all the microbes present. Because the smallerhydrocarbons can be transported across the cell membrane, the lightweight oil consuming microorganisms will grow faster than thoseconsuming high weight oil and will dominate the population that resultsfrom stimulation.

There are no methods in the art that effectively prevent the fasterbiodegradation of the light weight low-viscosity oil in comparison tothe slower biodegradation of the higher weight viscous oil in the mixedculture of a petroleum reservoir. There are reports of pure strains ofmicrobes that degrade only heavy oil (Purwasena I. A., et al. Proceedingof International Petroleum Technology Conference Doha, Qatar Dec. 7-9,2009). However, there is no method of preventing the growth ofindigenous short chain degrading microbes generally resident in mostreservoirs at less than 80° C.

Therefore, the same process that is beneficial to oil recovery is alsodetrimental to oil viscosity; and it is known that increasing theviscosity of the residual petroleum held within the reservoir willdecrease oil recovery.

Accordingly, there is a great need for new enhanced oil recoveryapproaches that are energy efficient, and can be reliably andsuccessfully used in large field situations to enable the recovery ofcurrently unrecoverable oil in existing oil fields. This new methodshould be able to selectively degrade certain target compounds found inthe oil remaining in the subject reservoir so that the oil will bemodified for better recovery by waterflood or by a chemical waterflood.Furthermore, the genes and the enzymes they code for can be modified andtheir expression regulated to best transform the oil for better recoveryand production. The host microorganisms should be selected so that theysurvive the extreme conditions in the reservoir at the time ofwaterflooding or during a chemical EOR waterflooding.

SUMMARY OF THE INVENTION

It is an object of this invention to provide microbes with genes formetabolic pathways that are useful for the enhanced recovery ofpetroleum oil from underground reservoirs, oil sands and other sourcesof heavy oil while suppressing the consumption of the lighter fractionof the petroleum. In addition, it is an object of this invention to givethe host or recipient organism of these genes a competitive advantagefor the specially modified environment of the hydrocarbon resourcereservoir before and or during an alkaline waterflood oil recoveryprocess.

In one aspect, the invention concerns a method of enhancing oil recoverycomprising

(a) inoculating an oil reservoir with a consortium comprising microbesthat are obligatory alkaliphilic, halo-alkaliphilic or alkaline tolerantand are deficient in their ability to utilize short chain hydrocarbonsof about 12 carbons or less but have the ability to convert hydrocarbonsinto fatty acids,

(b) allowing the consortium to proliferate and degrade hydrocarbons ofgreater than 12 carbons, and

(c) obtaining enhanced oil recovery from the oil reservoir.

In one embodiment, alkaline conditions are provided or are naturallypresent in the reservoir.

In a further embodiment, at least step (b) is performed under alkalineconditions.

In another embodiment, the consortium comprises microbes that arenaturally alkaliphilic, halo-alkaliphilic or alkaline tolerant.

In another embodiment, the consortium comprises microbes that areengineered to be alkaliphilic, halo-alkaliphilic or alkaline tolerant.

In yet another embodiment, the consortium comprises microbes that arenaturally deficient in their ability to degrade short chain hydrocarbonsof about 12 carbons or less.

In a further embodiment, the consortium comprises microbes that areengineered to be unable to degrade short chain hydrocarbons of about 12carbons or less.

In a still further embodiment, the consortium comprises microbes inwhich one or more metabolic pathways for degrading short chainhydrocarbons of about 12 carbons or less are down regulated or deletedor modified.

In a different embodiment, the consortium comprises microbes thatnaturally have the ability to degrade hydrocarbon chains of greater thanabout 12 carbons.

In another embodiment, the consortium comprises microbes that areengineered to be able to degrade hydrocarbon chains of greater thanabout 12 carbons.

In yet another embodiment, the consortium comprises microbes in whichone or more metabolic pathways for degrading hydrocarbon chains ofgreater than about 12 carbons are introduced.

In a further embodiment, the consortium comprises microbes that arecapable of utilizing high molecular weight hydrocarbons present in theoil reservoir as a carbon source.

In a still further embodiment, the consortium comprises microbes thatare capable of growing in a high salt environment (halophiles).

In an embodiment, high salt environment is provided or is naturallypresent in the reservoir.

In a still further embodiment, the consortium comprises microbes arecapable of utilizing simple carbons, where the simple carbons may, forexample, be selected from the group consisting of glucose, sucrose,mannose, starch, glycerin, organic acids, and other simple sugars.

In a different embodiment, in step (a) a nutrient mixture comprising asoluble carbon source is injected into said oil reservoir along with theconsortium, where the nutrient mixture may optionally further compriseat least one non-hydrocarbon nutrient, such as, for example, yeastextract, peptone, succinate, lactate, formate, acetate, propionate,glutamate, glycine, lysine, citrate, glucose, and/or vitamin solutions.

In a particular embodiment, the microbes are of the domain Archaeaand/or are bacteria.

In other embodiments, the consortium is able to grow at pH of 9.0 orhigher, or at pH of 10.0 or higher.

In various embodiments, the consortium may comprise microbes of thedomain Archaea and/or bacteria that are obligatory alkaliphiles,halo-alkaliphiles or alkaline tolerant, and have the ability to utilizering aromatic hydrocarbons and/or modified hydrocarbons containingsulfur and/or modified hydrocarbons containing nitrogen.

In another embodiment, the consortium comprises microbes of the domainArchaea and/or bacteria that are obligatory alkaliphiles,halo-alkaliphiles or alkaline tolerant, and have the ability to producesurfactants.

In still another embodiment, the consortium comprises microbes of thedomain Archaea and/or bacteria that are obligatory alkaliphiles,halo-alkaliphiles or alkaline tolerant, and that have been engineered toproduce surfactant. In one other embodiment, the production ofsurfactant is independent of the expression of genes that code for thedegradation of hydrocarbons.

In yet another embodiment, the consortium comprises microbes of thedomain Archaea and/or bacteria that are obligatory alkaliphiles,halo-alkaliphiles or alkaline tolerant, and have the ability to produceextracellular polymers.

In a further embodiment, the method of the present invention furthercomprises the step of waterflooding said reservoir with an alkalinefluid or a fluid containing a compound toxic to indigenous microbes toreduce the concentration of microbes that have the ability to utilizeshort chain hydrocarbons of about 12 carbons or less.

In a still further embodiment, the method of the present inventionfurther comprises the step of adding to the reservoir at least onechemical inhibitor to control a metabolic pathway of at least oneindigenous microbe present in the reservoir and/or obligatoryalkaliphilic, halo-alkaliphilic or alkaline tolerant microbe inoculatedinto the reservoir.

In an embodiment, the chemical inhibitor inhibits the degradation ofshort-chain alkanes by said indigenous and/or inoculated microbe.

In another aspect, the invention concerns an isolated microorganism,preferably of the domain Archaea or bacteria, that (i) is an obligatoryalkaliphile, halo-alkaliphile or alkaline tolerant, and (ii) isdeficient in its ability to degrade short chain hydrocarbons of about 12carbons or less.

In one embodiment, the microorganism is obligatory alkaliphilic,halo-alkaliphilic or alkaline tolerant and is deficient in their abilityto utilize short chain hydrocarbons of about 12 carbons or less but hasthe ability to convert hydrocarbons into fatty acids.

The microorganism may naturally have the described properties and/or maybe engineered to possess one or more of the described properties.

In various embodiments, the microorganism is able to grow in alkalinityof pH 9.0 or higher, or in alkalinity of pH10.0 or higher.

In another embodiment, the microorganism has the ability to utilizehydrocarbon chains of greater than 12 carbons.

In a further embodiment, the microorganism has the ability to utilizemodified hydrocarbons containing sulfur.

In a still further embodiment, the microorganism has the ability toutilize modified hydrocarbons containing nitrogen.

The invention also concerns a consortium comprising microbes thatpossess one or more of the properties described above.

The invention further concerns a consortium comprising one or more typesof microorganisms as hereinabove described.

In a further aspect, the invention concerns an oil reservoir, comprisinga consortium herein.

The consortium used in the methods herein may contain one or moredifferent types of microorganisms. Thus, for example, it may containmicroorganisms that are both obligatory alkaliphilic, halo-alkaliphilicor alkaline tolerant and halophiles, in addition to being deficient intheir ability to utilize short chain hydrocarbons. It is also possible,however, that the consortium contains two different types ofmicroorganisms, one being obligatory alkaliphilic, halo-alkaliphilic oralkaline tolerant and deficient in their ability to utilize short chainhydrocarbons, and the other one being a halophile which is alsodeficient in its ability to utilize short chain hydrocarbons.

It is noted that two or more of the various embodiments listed above orotherwise disclosed herein can be used in any combination, and any andall of such combinations are within the scope of the present invention.

It is further noted that various embodiments described in connectionwith one aspect of the invention are also contemplated with respect toother aspects of the invention. Thus, embodiments described withreference to the methods of the present invention also apply to otheraspects, such as the consortium of microorganisms or the isolatedmicroorganisms present in such consortium, as applicable.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of the patent application publication with colordrawing(s) will be provided by the Office upon request and payment ofthe necessary fee.

FIG. 1 illustrates a pathway for the degradation of alkanes bymicroorganisms.

FIG. 2 illustrates the conversion of fatty acids to soap at alkaline pH.

FIG. 3 illustrates the emulsification of oil by fatty acid soaps atalkaline pH.

FIG. 4 shows the alignment of the amino acid sequences of the LadA longchain alkane monooxygenase (SEQ ID NO: 1) with the hypotheical proteinGen ID 9420269 HacjB3_12265 from Halalkalicoccus jeotgali B3alkalitolerant halophiles (SEQ ID NO: 2) and with another hypotheticalprotein from the halophile, Halorubrum lacusprofundi (ATCC 49239) GeneID 7401614 Hlac 0096 (SEQ ID NO: 3).

FIG. 5 Generation of acd3 and aldY5 gene knock outs of Haloferaxvolcanii DS2. The 1.3 kilobase PCR band generated from the genomes ofmultiple mutant isolates, but not from that of wildtype Haloferaxvolcanii, demonstrates successful acd3 and aldY5 gene knock outs.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

Unless otherwise defined, all terms of art, notations and otherscientific terminology used herein are intended to have the meaningscommonly understood by those of skill in the art to which this inventionpertains. In some cases, terms with commonly understood meanings aredefined herein for clarity and/or for ready reference, and the inclusionof such definitions herein should not necessarily be construed torepresent a substantial difference over what is generally understood inthe art. The techniques and procedures described or referenced hereinare generally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized molecular cloning methodologies described in Sambrook etal., Molecular Cloning: A Laboratory Manual 2nd. Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Also, forexample, Current Protocols in Molecular Biology, Supplement 93, January2011, John Wiley & Sons, Inc. As appropriate, procedures involving theuse of commercially available kits and reagents are generally carriedout in accordance with manufacturer defined protocols and/or parametersunless otherwise noted.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise.

Throughout this specification and claims, the word “comprise,” orvariations such as “comprises” or “comprising,” will be understood toimply the inclusion of a stated integer or group of integers but not theexclusion of any other integer or group of integers.

All publications mentioned herein are incorporated herein by referenceto disclose and describe the methods and/or materials in connection withwhich the publications are cited. Publications cited herein are citedfor their disclosure prior to the filing date of the presentapplication. Nothing here is to be construed as an admission that theinventors are not entitled to antedate the publications by virtue of anearlier priority date or prior date of invention. Further, the actualpublication dates may be different from those shown and requireindependent verification.

The term “oil reservoir” is used herein in the broadest sense andincludes all forms of hydrocarbon deposits, including, withoutlimitation, producing wells, non-producing wells, experimental wells,exploratory wells, and the like, which may be accessible by any means,such as, for example, one or more wellbores.

The term “crude oil” refers to a naturally occurring, flammable liquidfound in rock formations and comprises a complex mixture of hydrocarbonsof various molecular weights, plus other organic compounds. Withoutlimitation, the crude oil may contain, for example, a mixture ofparaffins, aromatics, asphaltenes, aliphatic, aromatic, cyclic,polycyclic and/or polyaromatic hydrocarbons. The crude oil may begeneric or may be from a reservoir targeted for enhanced oil recovery inaccordance with the present invention.

The terms “well” and “reservoir” may be used herein interchangeably andrefer to a subterranean or seabed formation from which oil may berecovered. The terms well and reservoir include the physical/chemicalcomposition of the soil-rock-sediment structure of the reservoir belowthe surface.

The term “environmental sample” means any substance exposed tohydrocarbons, including a mixture of water and oil comprisingmicroorganisms. As used herein, environmental samples include water andoil samples that comprise indigenous microorganisms and/or populationsof microorganisms of varying genus and species. The environmentalsamples may comprise a microbial consortium unique to a geographicregion or target reservoir, or, alternatively the microbial consortiummay be adaptable to other environment sites, geographies and reservoirs.

The term “microbe” is used herein in the broadest sense and includes allmicroorganisms, including bacteria, fungi, archae, and protists, andmicroscopic animals, such as plankton, planarian and amoeba. Preferredmicrobes for the purpose of the present invention are bacteria andarchae.

The term “microbial consortium” is used herein to refer to multipleinteracting microbial populations. Members of a consortium communicatewith one another. Whether by trading metabolites or by exchangingdedicated molecular signals, each population or individual detects andresponds to the presence of others in the consortium. This communicationenables a division of labor within the consortium. The overall output ofthe consortium rests on a combination of tasks performed by constituentindividuals or sub-populations.

Archaea comprise one of the three distinct domains of life, withbacteria and eukaryotes. For a review, see, e.g. Makarove and Koonin,Genome Biology 4:115 (2003).

The term “alkali tolerant” is used herein to refer to an organismcapable of growing at pH of 9 or more, but with optimum growth rates atneutral pH.

The term “obligatory alkaliphile” (also spelled alkalophile) is usedherein to refer to an organism whose growth is optimal in at least twopH units above neutrality or a pH of 9 or more.

The term “obligatory haloalkaliphile” is used herein to refer to anorganism whose growth is optimal at a salt concentration of 100,000 ppmor more and at a pH of 9.0 or more.

For a review of alkaliphilic and alkalitolerant microorganisms see, e.g.Koki Horikoshi, Microbiology and Molecular Biology Reviews, December1999, p. 735-750. And “Alkaliphiles” 1999 ISBN 90-5702-458-6 publishedby Kodanha Ltd Tokyo Japan, author Koki Horikoshi.

The term “halophile” is used herein to refer to an extremophile thatthrives in environments with very high concentrations, typically atleast about 5% (50,000 ppm), or at least about 10%, or at least about15% of salt.

The term “obligatory halophile” is used herein to refer to anextremophile whose growth is obligately dependent on high saltconcentrations, typically at least about 5% (50,000 ppm), or at leastabout 10%, or at least about 15% of salt.

The terms “repression” and “inhibition” with reference to geneexpression are used herein interchangeably and refer to any processwhich results in a decrease in production of a gene product, regardlessof the underlying mechanism. A gene product can be either RNA orprotein. Gene repression includes processes which decrease transcriptionof a gene and/or translation of mRNA. Thus, specifically included inthis definition are processes that inhibit the formation of atranscription initiation complex along with those that decreasetranscription rates and those that antagonize transcriptional activationis gene repression. These repressions can be either reversible orirreversible, both of which are specifically included in thisdefinition. The repression can be partial with only lower levels of thegene expressed or complete with no genes expressed and no protein geneproduct synthesized.

The term “lateral gene transfer” is used herein in the broadest senseand refers to the transmission of genetic information from one genome toanother.

The term “surfactant” as used herein means microbially producedsurface-active agents, including, but not limited to, glycolipids (e.g.sophorose lipid or rhamnose lipid), lipoproteins, polysaccharide-fattyacid complexes, mono- and diglycerides, lipoheteropolysaccharides,peptidolipids, neutral lipids, corynomycolic acids, trehalosedimycolates and polysaccharide-protein complexes.

The term “soap” is used herein in the broadest sense to describe anyhydrocarbon with one or more carboxyl groups, or also referred to as afatty acid, that has been converted to the acid salt by alkaline removalof the hydrogen ion and replacement with a mono or divalent metal ion.

The term “hydrocarbon” is used herein in the broadest sense to describeany organic compound that contains only carbon and hydrogen (in somecases may contain sulfur or nitrogen atoms). The term specificallyincludes, without limitation, saturated hydrocarbons (alkanes),unsaturated hydrocarbons (including alkenes and alkynes), cycloalkanes,and aromatic hydrocarbons (arenes).

The term “short chain hydrocarbon” is used herein to mean both straitchain alkanes or branched chain alkanes containing 12 carbons or less.

A “short chained alkane”, as defined herein, contains 1 to 4 carbonatoms.

A “high molecular weight hydrocarbon”, as defined herein, is ahydrocarbon having at least about 40 carbons, for example, a hydrocarbonhaving between about 40 and about 60, or between about 40 and about 80,or between about 40 and about 100, or between about 40 and about 120carbons.

The term “waterflooding” is used herein to refer to a crude oil recoveryprocess wherein a fluid containing water is pumped into an injectionwell in contact with an underground formation containing crude oil todrive the residual crude oil towards another well also in contact withan underground formation for the purpose of producing oil.

The term “nutrient supplementation” refers to the addition of nutrientsthat benefit the growth of microorganisms that are capable of usingcrude oil as their main carbon source but grow optimally with othernon-hydrocarbon nutrients, i.e., yeast extract, peptone, succinate,lactate, formate, acetate, propionate, glutamate, glycine, lysine,citrate, glucose, and/or vitamin solutions.

II. Detailed Description

The present invention concerns enhanced methods for microbial oilrecovery, characterized by the use of microbes that are obligatoryalkaliphilic, halo-alkaliphilic or alkaline tolerant and additionallyare deficient in their ability to utilize short chain hydrocarbons whilehaving the ability to convert hydrocarbons into fatty acids. The latterproperty is useful for emulsifying the oil at alkaline pH.

In certain embodiments of the present invention, means are provided toutilize an alkaline microbial waterflooding process to mobilize oilremaining in a petroleum reservoir. In this microbial process the acidcontent of the crude oil is increased by microbial degradation of thehydrocarbons at alkaline pH. Microorganisms preferred for this processare selected or engineered to convert oil hydrocarbon components toalcohols, aldehydes, ketones and/or fatty acids that are the most usefulat alkaline pH to aid in the emulsification and dispersion of oil intothe waterflood drive fluid. At alkaline pH the preferred size range offatty acids are converted to soaps which can reside at the oil to waterinterface. Smaller hydrocarbons are converted to only alcohols oraldehydes which do not have a negatively charged carboxyl group such asfatty acids. These low molecular weight alcohols of about two to eightcarbons are more useful as co-surfactants than the corresponding lowmolecular weight fatty acids. The methods provided by the presentinvention are designed to select the preferred size range ofhydrocarbons modified by the microbial consortium and direct thepathways to produce the products that are most beneficial for oilemulsification and reduction of interfacial tension.

According to the present invention, certain metabolic pathways such aspathway(s) required for complete degradation of short chain alkanes areselected out of microorganisms present in a consortium. As a result, theconsortium will contain microbes that are not only obligatoryalkaliphilic, halo-alkaliphilic or alkaline tolerant, but are alsodeficient in their ability to utilize short chain hydrocarbons (e.g.less than about 20 carbons, or about 12 carbons or less).

In particular, bacteria and other microbes (e.g. Archaea) are carefullyselected or modified to be deficient in their ability to consume thelower weight hydrocarbons all the way through the β-oxidation pathway.Fatty acids that enter the β-oxidation pathway by conversion to Acyl-CoAcan be converted to energy for cell growth. A method of engineering apathway into E. coli for production of fatty acids and fatty alcoholsand other bio-fuels is described by Eric J. Steen et al. in “Microbialproduction of fatty-acid-derived fuels and chemicals from plant biomass”Nature 463,559-562 (28 Jan. 2010) doi:10.1038/nature08721: and isincorporated by reference. The referenced report is an example ofknocking out a gene for β-oxidation of fatty acids for the purpose ofproducing an accumulation of fatty acids and alcohols for bio-fuelproduction. Fatty acids in a range of C8-C18 are reported inconcentrations as high as 0.3 g/l.

The preferred products of bioconversion are short chain alcohols whichare useful in reducing the viscosity of oil and functioning as aco-surfactant in emulsifying oil. A consortium of microbes is selected,modified or controlled so that the microbial culture relies on either asoluble carbon source provided by the nutrient mixture injected with themicrobes and water flood or the bio-consumption of high molecular weighthydrocarbons present in the petroleum. The metabolic pathways thatdegrade only the higher molecular weight hydrocarbons are notdeliberately down regulated or deleted. These pathways are beneficialbecause the consumption of higher molecular weight carbohydratesprovides an additional carbon source, and the removal of high molecularweight oil reduces the viscosity of the oil. Reduction of viscosityimproves both the value and the recovery of the petroleum.

In addition, the conversion of hydrocarbons in the range of C-13 to C-40to fatty acids is useful for the emulsification of oil at alkaline pH.This range of fatty acids is small enough to be extracted from the oildroplet and react with the alkaline material to form soap by loss of aproton. The preferred size range of the fatty acids can be refined forthe temperature, salinity and other environmental factors anticipated inthe reservoir. This refinement can be made by laboratory experimentationby addition of various fatty acids such as hexadecanoic acid toreservoir petroleum oil samples (as described in Example 3) andmeasuring interfacial tension with the drive fluid. In addition todetermining the preferred size range by a determination of IFT reductionof oil and drive fluid, oil with various levels (acid number defined asmg of KOH per gram of oil needed to neutralize the acid) and types(number and configuration of carbons atoms) of fatty acids can beanalyzed for recovery in sand pack columns or core flood laboratoryexperiments. The advantage of these experiments is that they can be usedto quickly determine the size range for each type of fatty acid or fattyalcohol that will be most beneficial for emulsifying the type of oilfound is various reservoirs. Also the pH and salt concentration can beevaluated for the drive fluid to be used in field oil recovery process.

These size ranges for each type of hydrocarbon, that will yield the mosteffective increase in oil recovery at the conditions anticipated in theactual oil field application, can then be used to select metabolicpathways for placement and expression of genes for the needed enzymes inthe host engineered microorganism. The engineered host microorganismscan then be tested in the laboratory to determine if they can grow onthe type of oil found in the target reservoir and increase the fattyacid content of the oil as it degrades it. The conversion of oilsubstrate molecules into fatty acids and fatty alcohols can bedetermined by gas chromatography of the oil, liquid chromatography ofthe aqueous phase or by basic aqueous extraction of the fatty acids fromthe oil followed by making a derivative for gas chromatography. Directmass spectrometry or in combination with gas or liquid chromatography isalso a possible way to analyze and quantitate the fatty acids andalcohols formed by microbial degradation of the petroleum. By thesemethods, each strain of engineered microbe can be evaluated for itsability to transform the oil into an acid number that is higher than thenative oil.

Not only will the total acid number be increased, but the size range ofacids can be made of the size that is best for decreasing IFT betweenthe oil and drive fluid. Small acid products that are too hydrophilic toreside at the oil surface would move into the hydrophilic drive fluid,and would not be useful for oil recovery. In addition they might bedetrimental by reducing the pH of the drive fluid. At the other extreme,very large hydrocarbons may be so hydrophobic that they would resideentirely in the oil phase and not effectively contribute to theextractable acid number or help in lowering the IFT of the oil.Therefore, the oil that is degraded and transformed into acids will bevery useful for improving oil emulsification at alkaline conditions.Therefore, the total oil remaining will become a better target foralkaline flooding or alkaline surfactant polymer flooding.

The oil recovery process wherein the drive fluid is maintained at analkaline pH is referred to as alkaline flooding. The oil recoveryprocess wherein the drive fluid also contains a chemical polymer and achemical surfactant in combination with and alkaline drive fluid isreferred to as alkaline surfactant polymer waterflooding or ASPwaterflooding. The aqueous drive solution is an alkaline chemical, suchas sodium hydroxide, carbonate or bicarbonate. The alkaline chemicalreacts with the acid compounds in the crude oil and produces thesurfactant in situ. IFT reduction is the main mechanism of oilemulsification. The process is complex due to various reactions with thereservoir rock and fluids. Adverse interactions with the reservoir rockcan be minimized by use of moderate pH alkaline chemicals, such assodium carbonate and bicarbonate. When this process is combined with theadditional chemical formulations of an added chemical surfactant andchemical polymer (ASP) the major recovery mechanisms are both IFTreduction and improvement in mobility ratio. Both methods require thatthe reservoir oil have an acid number of 0.5 or more to be economicallyeffective. A more detailed description of alkaline flooding for EOR wasreported by J. Xie, B. Chung, L. Leung at the Society of PetroleumEngineers International Thermal Operations and Heavy Oil Symposium heldin Calgary, Alberta, Canada, 20-23 Oct. 2008 and is available from theSPE in publication SPE/PS/CHOA 117221 PS2008-323.

In addition to transforming the native oil into one of higher acidcontent, especially of the type of acids (soluble to some degree in boththe oil and the water phase and having both a lipophilic and ahydrophilic region) that are best suited for soap formation during analkaline flooding or ASP flooding, another embodiment is thesimultaneous increase of the acid content of native oil while analkaline process is ongoing. During the alkaline process, thealkaliphile microbes grow on the oil surface and produce the range offatty acids that aid the emulsification of oil. Because this happens onthe other surface of the oil liquid, small droplets of oil can form andbe emulsified into the alkaline water. This process then exposes a newoil surface to the growing microbes. In this way the alkaliphiles canbore into the oil faster as the oil droplets are removed by the alkalinewater. The faster the microbes are at converting hydrocarbons into fattyacids the faster they are at emulsifying the entire oil layer. Thereforea microbe that can grow rapidly at alkaline pH will recover more oil andat a faster rate than a microbe that must convert hydrocarbons to fattyacid at neutral pH to be followed by an alkaline waterflood.

In addition to the facilitation of emulsifying soaps from the fattyacids at alkaline pH, the high pH also acts to kill off or suppress thegrowth of indigenous microbes. Microorganisms that have adapted toextreme environments such as high pH and high concentrations of saltoften have evolved with significant changes to their protein amino acidsequences. Alkaliphiles and haloalkaliphiles enzymes are significantlyhigher in negatively charged amino acids and low in the positivelycharged amino acid lysine, which helps prevent the acquisition of genesfor light chain oil utilization enzymes.

If the inoculating microbes lacked certain unwanted genes, but theinoculating microbes were similar to the indigenous microorganisms, theycould pick up unwanted genes from the indigenous microorganisms. Even ifthe engineered culture was robust and quick growing, it could acquiregenes that coded for the metabolism of light weight oil. This couldoccur by the process of Lateral Gene Transfer (LGT), which is known tooccur in many natural environments. Picking up such genes would give themicrobes a competitive or evolutionary advantage, and they would soondominate the population.

By the use of alkaline fluids and microbes that prefer alkalineconditions, this invention provides a means for preventing LGT so thatthe engineered or selected culture of microbes with the desiredproperties does not acquire unwanted or detrimental pathways forimproved oil production. Lateral gene transfer is common among bacteriaand Archaea as a mechanism of genetic information sharing betweendifferent species. It is believed to play a significant role inevolution and is also known to occur in higher organisms. The preventionof LGT is an important part of the successful implementation ofengineered microorganisms.

By preventing the metabolism of light oil, the corresponding viscosityincrease, caused by the removal of the light oil fractions, is alsoprevented. As stated above, the higher the viscosity of oil, the lowerthe recovery will be. The beneficial effects of the microbes such asreduction of IFT, increase sweep efficiency and improved mobility ratiocould be negated by increase in viscosity. However, if the microbes canonly consume heavy oil or other carbon sources, the major detrimentaleffect can be avoided. This makes the MEOR process of the presentinvention more predictable and more effective.

The use of an obligate alkaliphile or halo-alkaliphile or alkalinetolerant microorganism requires that certain genes taken from anon-alkaliphile or neutral pH microbe be removed or modified to changethe amino acid sequence of any proteins they code for. These changes areneeded to render the proteins functional at a higher pH. These requiredchanges can be determined by analysis of homologous proteins found inboth neutral pH and alkaline pH microorganisms. In addition, threedimensional structural analyses can be used to determine surfacepositively charged resides such as lysine which may be changed tonegatively charged amino acid residues such as aspartic acid. This typeof amino acid residue changes will result in an increase negativecharge, which is generally beneficial to high salt and high pHfunctionality. A large number of potentially beneficial changes can bemade and then tested by expression in a model alkaliphile orhalo-alkaliphile such as Natronobacterium magadii and N. gregoryi.

The study of hydrocarbon bio-degradation provides an understanding ofthe mechanism of short chain alkane metabolism. The shorter chainalkanes are made soluble in water generally with the aid of surfactantsproduced by the bacteria or Archaea. Then, the soluble alkane adsorbsonto the cell's hydrophobic membrane and is transported across themembrane of the microbe. Enzymes at the membrane convert the alkane toan alcohol. Subsequent chemical reactions catalyzed by other enzymesconvert the alcohol to an aldehyde and then to an organic acid, alsoreferred to as a fatty acid. The fatty acid can then be furthermetabolized by the cell for energy and carbon building blocks for itsgrowth. This biology of the short chain alkane metabolism is the moststudied and the best understood. Metabolism of larger or highermolecular weight hydrocarbon is more complex and less well understood.The larger or higher weight hydrocarbons are much less soluble and moredifficult to transfer across the cell membrane. However, thebiodegradation of high molecular weight oil is known to occur, however,it happens at a slower rate. A more detailed description of biologicaldegradation of hydrocarbons is reviewed by J. D. Van Hamme, A. Singh,and O. Ward in Microbiology and Molecular Biology Reviews, December2003, p. 503-549, Vol. 67 No 4, DOI: 10.1128/MMBR.67.4.503-549.2003.This invention relies on retarding the light chain alkane metabolism bythe engineered or selected microbes. The preferred modification is tostop the conversion of alkanes in the range of two to eight carbons atthe corresponding two to eight carbon alcohol and to transfer it to theoil to water interface where it can function to reduce the viscosity ofthe oil and act as a co-surfactant.

In certain embodiments of the methods of the present invention, genesthat code proteins in the alkane hydroxylase pathway, which are capableof degrading light weight and low viscosity hydrocarbons, are inhibited,e.g., deleted, mutated or down regulated in the selected or engineeredmicrobe. In addition, LGT from the environment is prevented because theindigenous enzymes are less active at the higher pH. That is, theacquisition of similar genes that code for degradation of light weightoil from other neutral pH microbes are unlikely to be functional at thehigh pH of the alkaline microbes. However, the production ofsurfactants, co-surfactants or other metabolites, beneficial to oilmobilization, is not prevented. The expression of genes needed for theproduction of surfactants is maintained without the consumption of lowviscosity oil.

Different sets of genes code for each of the different metabolicpathways that make it possible for microbes to feed on hydrocarbons asan energy source. The degradation and consumption of the highermolecular weight hydrocarbons is generally enabled by different genesthen those that code for the light chain metabolic pathways. Genes thatcode for the enzymes required for hydrocarbon degradation and the genesfor surfactant production may be regulated by the same promoters.However, with current molecular biology technology it is possible tomove and separate each set of genes so that they can be independentlycontrolled.

The enzymes that degrade hydrocarbons have different substratespecificities. The first step in the degradation of alkanes is theoxidation of either the terminal carbon or an internal carbon to form aprimary or secondary alcohol. The monooxygenases are one type of enzymethat catalyze the first step in the metabolism of hydrocarbons and havebinding sites that show a preference or specificity for differentlengths of straight chain alkanes. In addition, there are monooxygenasesthat will oxidize aromatic hydrocarbons of different sizes. Many of thegenes have been isolated and their sequences characterized. Many othershave not yet been isolated, but are expected to have similar sequencesand different specificities. With probes for genes based on highlyconserved regions of protein sequences of key enzymes and proteinsequences that determine substrate specificity, new genetic informationcan be obtained from microbes inhabiting petroleum sites.

Currently, there is nucleotide and amino acid sequence informationavailable for many monooxygenases that degrade different sizes and typesof petroleum hydrocarbons that highly conserved regions have beenidentified. Highly conserved regions of protein sequences have beenidentified that are required for catalytic activity. Others aresubstrate specific and will vary with the size and type of hydrocarbonthat they oxidize. For example, the 8 histidine amino acid residues thatare required for catalytic activity in all alkane monooxygenases are inthree histidine boxes (Hist1, HE[L/M]XHK (SEQ ID NO: 8); Hist2, EHXXGHH(SEQ ID NO: 9); and Hist3, LQRH[S/A]DHHA (SEQ ID NO: 10)) reported by J.B. van Beilen et al in Applied and Environmental Microbiology, December2002, p. 5933-5942, Vol. 68, No. 12, DOI10.1128/AEM.68.12.5933-5942.2002. This knowledge can be used to searchfor microbes within an environment that can degrade varioushydrocarbons. Metagenomic sequencing of the environmental samples can becarried out, and new oxygenase genes identified by DNA sequencehomology. Probes to the highly conserved sites can be used to isolategenes that code for monooxygenases that exist within the microbesinhabiting alkaline and petroleum containing sites. For example,alkaliphilic and halophilic hydrocarbon-utilizing microbes were isolatedfrom oily alkaline intertidal areas of the Kuwaiti coasts by H.Al-Awadhi et al. in Applied Microbiol Biotechnology (2007) 77:183-186.Further analysis based on sequences of the substrate specific sites canidentify genes that code for higher molecular weight hydrocarbonutilization at high pH.

Although slower than light chain utilization, degradation of heavy chainhydrocarbons can provide a supplemental carbon source without detrimentto the viscosity or the value of the petroleum oil. Microbes have beenisolated that can grow only on heavy petroleum components. These havebeen shown to have genes that code for enzymes that are specific forcertain heavier hydrocarbons and to lack the genes for utilization ofthe lighter short chain alkanes. For example, L. Wang et al. reportedisolating Geobacillus thermodenitrificans NG80-2 from a deepsubterranean oil reservoir in northern China that degrades andmetabolizes only long chain (C15-C36) n-alkanes, but not short-chain(C8-C14) n-alkanes. The complete genome sequence of G.thermodenitrificans NG80-2 has been deposited in the GenBank databaseand is incorporated with the corresponding publication in Proc Natl AcadSci USA, Mar. 27, 2007 p. 5602-5607 by reference. Comparison of proteinsequences can be done to identify specific substrate sequences andprobes can be made to the genes for either short or long chain alkanemonooxygenases. These probes can be used to screen DNA isolated from aspecific site or oil reservoir. By this method and other methods ofmicrobiology, the microbes that are responsible degradation of heavy andlight oil in a reservoir can be identified. For example, by searchingGenBank with the protein sequence of LadA, a long alkane monooxygenase,from the thermopile and non-halophile, Geobacillus thermodenitrificansNG80-2, a protein sequence with 49% identity to LadA with most of theamino acids resides at the putative reactive site and flavin bindingsite identical to the amino acid residues found in Halalkalicoccusjeotgali (DSMZ 4425), which is a alkaliphile and a halophile. Anotherhypothetical protein sequence from the genome of Halorubrumlacusprofundi (ATCC 49239), which is reported to be a halophile but notan alkaliphile, is also 49% identical in protein sequence and nearlyidentical at the putative active pocket to the monooxygenase found in G.thermodenitrificans NG80-2. By comparing sequences of homologousproteins from alkaline pH environments to neutral environments,predictions can be made for changes to surface amino acid residues thatwill adapt the proteins for higher pH optimums. This type of sequenceinformation can be used to engineer amino acid changes to make neutralpH and low salt enzymes functional at either high pH or high salt andhigh pH. Each of the modified sequences can be expressed as new enzymesthat can be analyzed for its pH optimum and salt solubility.

In addition to high molecular weight hydrocarbons, petroleum oilcontains compounds that are not desirable to have in oil that will berefined into various petroleum products. One major group of undesirablecompounds is modified hydrocarbons high in sulfur. Sulfur can be thethird most abundant element in crude oil and is especially high in heavyoil. Lowering the sulfur content would increase the value of the crudeoil. Bacteria that are capable of selectively attacking the C—S bondshave been isolated and their metabolic pathways elucidated. Most strainsstudied have been aerobically grown and include; Rhodococcuserythropolis, Nocardia spp., Agrobacterium sp. Strain MC501,Mycobacterium spp., Gordona sp. Strain CYKS1, Klebsiella spp.,Xanthomonas spp., and the thermophile Paenibacillus. These bacteria havebeen shown to be effective at desulfurization of various sulfurcontaining hydrocarbons found in crude oil. However, the process is atwo phase oil and water system that requires surfactants andenergy-intensive mixing. To achieve a sulfur removal rate of over 50%high water to oil ratios were needed in well mixed and aerated reactors.Important aspects of the process include reactor design, productrecovery and oil-water separation.

Another group of undesirable hydrocarbons are nitrogenous compounds.Crude oil can contain about 0.5% to 2.1% nitrogen with 70% or more aspyrroles, indoles and carbazole nonbasic compounds. These compounds arepoisons to cracking catalysts, toxic and result in air pollution.Removal of the nitrogenous compounds would increase the value of oilrecovered by the MEOR process. Several species of bacteria have beenisolated that contain metabolic pathways for the oxidativetransformation of nitrogenous compounds found in crude oil. A review ofthese bacterial processes was published by Kaiser, J. P. et al. inMicrobiol. Rev. 60:483-498. The genes responsible for carbazoledegradation by Pseudomonas sp. strain CA10 were identified and clonedinto E. coli by Sato et al and were reported to transform a wide rangeof aromatic compounds. The results are published in J. Bacteriol. 179:4841-4849 in 1997.

The alkaline adaptation of enzymes needed for the desulfurizationpathways or denitrogenation pathways can be done make the proteinsfunctional s in the high pH environment of the host alkaliphile. Theirincorporation into a culture designed for oil recovery can also reducethe sulfur or the nitrogen content of the recovered oil. However,because these are oxidative processes, it is important that genesresponsible for light chain metabolism be eliminated so that the shortchain alkanes are not rapidly degraded.

Many microbes that utilize hydrocarbons can also utilize simple andsoluble carbon sources. Generally, when the concentration of a simplecarbon source is high enough, the expression of all the genes needed forthe utilization of hydrocarbons is down regulated. Some examples ofsoluble carbon sources comprise simple sugars, glycerin, starch, fattyacids and other organic molecules. This is one mode of preventing shortchain alkane utilization as long as simple carbon sources are maintainedat a high concentration. If the host or recipient microbe, engineeredfor the oil reservoir environment, does not contain adequate pathwaysfor the utilization of inexpensive soluble carbon sources, genesrequired for those pathways could be transferred into the host microbe.

That is, by providing a soluble carbon and energy source at sufficientlevels to maintain living cells and cell growth, the indigenous microbesmay become nondependent on alkane hydrocarbon metabolism for growth andsurvival. This could lead to down-regulation and low expression of genesor even loss of the genes that code for enzymes that make usefulmetabolites such as surfactants that emulsify the insolublehydrocarbons.

Therefore, a means for maintaining high expression of genes and levelsof certain proteins must be provided. This can be done by a number ofmolecular biology techniques, including, but not limited to, placing thegenes coding for each of the metabolic products such as surfactantproduction under the control of an inducible or constitutive promoter.This will allow for high expression by both transcription andtranslation of these genes. This provides means for preventing a downregulation that can occur with the wild type promoter when the celldetects a high level of easier to metabolize or preferred carbon source.In conventional MEOR processes that use only naturally occurringcultures of oil consuming microbes in combination with indigenousmicrobes present in the oil reservoir, the addition of too much of asimple carbon source, such as molasses, could lead to a reduction ofsurfactant production and unexpectedly lower oil emulsification.

The problem with relying on naturally occurring microbial processes isthat they become less effective at both oil degrading and oil recoverywhen they are supplied with an easily metabolized carbon and energysource, such as molasses. However, not supplying any simple carbonsource could slow growth and also lead to low oil production. Inaddition, the lack of a supplied carbon source will select for thestrains of microbes that can utilize the hydrocarbons that exist withinthe oil reservoir. Furthermore, microbes that have genes that enablethem to consume light weight oil will grow and multiply faster than anymicrobe, added or indigenous, that only contains genes for heavy oilconsumption. Therefore, it is best to provide adequate carbon sourcesfor the engineered or selected strains so that they can grow fast enoughto prevail over the indigenous strains that have the ability tometabolize short chain alkanes.

Gene promoters contain specific DNA sequences and response elements thatare recognized by proteins known as transcription factors. These factorsbind to the promoter sequences recruiting RNA polymerase, the enzymethat copies or transcribes the gene coded for in the DNA into amessenger RNA (mRNA). The mRNA can then migrate to a ribosome where itis translated into a protein or gene product.

Gene repression and inhibition of expression refer to any process, whichresults in a decrease in production of a gene product, whether by areversible or an irreversible process. A gene product can be either RNAor protein. Gene repression includes processes which decreasetranscription of a gene and/or translation of mRNA. For example, aprocess that inhibits the formation of a transcription initiationcomplex or those that decrease transcription rates or those thatantagonize transcriptional activation is gene repression.

An inducible promoter is one that is controlled or regulated by someextracellular factor that can increase or decrease the transcription andtranslation of genes into their products. In a specific example ofn-alkane degradation, the alk genes of Pseudomonas oleovorans areresponsible for the degradation of n-alkanes. These genes are located intwo gene clusters that are controlled by a promoter which is controlledby the AlkS protein. This protein is responsive to the hydrocarbonoctane. The presence of octane will increase or activate the expressionof these genes and their protein products. However, this same promoteris also down-regulated or repressed by the presence of a preferredcarbon source such as organic acids. These bacteria would both emulsifyand degrade n-alkanes unless high levels of a preferred carbon sourceare supplied. In this case, the genes for hydrocarbon degradation wouldbe turned off. This would limit the usefulness of microbes inremediating hydrocarbon contaminated sites and could become lesseffective at degrading hydrocarbons if given more easily metabolizedcarbon sources. However, by inactivating the down-regulation of thepromoter by preferred carbon sources, and inactivation of genes from thecluster that are needed for the metabolism of alkanes, this microbe canbe engineered into an oil emulsifying bacterium that can grow on solublecarbon sources.

One means for inactivating the down-regulation by a simple solublecarbon source is to mutate the sequence of the AlkS protein that bindsthe carbon source in such a way as to not affect the octane bindingsite. Another method is to engineer the genes coding for the surfactantor bio-polymer production pathway to be under the control of a differentpromoter. This provides a way of controlling the production ofsurfactant or bio-polymer, independent of carbon source.

By maintaining growth on a medium containing a soluble carbon source,the genes that code for short chain alkane hydrocarbon metabolism can byinactivated by a number of means. Methods suitable for inactivation ofthese genes include, but are not limited to, chemical mutagens and UVand other forms of radiation. In addition, functional genes can bereplaced by nonfunctional genes. The technology of gene silencer,developed by A. Fire et al., Nature 391(6669):806-11 (1998), has lead toa better understanding of how genes regulate mammalian cell function.These methods of inactivating specific genes can be used to locate keygenes responsible for any metabolic process that a cell or microbe cancarry out. In addition, if selected microbes for improved oil do nothave completed genomic sequences available in the public domain, theentire genome can be sequenced rapidly by current technology at a fairlylow cost.

One functional gene may be used to replace another functional gene. Thenew gene may also include a reporter gene for easy selection of microbescontaining the new gene. For example, a functional cluster of genes thatcode for a high molecular weight hydrocarbon metabolism pathway may beinserted into a host cell. It may replace a gene cluster for lowermolecular weight hydrocarbon metabolism pathway that has been removed orinactivated. In addition, the cell may be given a resistance gene for anantibiotic or other toxin. This is a commonly used method for selectingcells that have successfully incorporated new genetic material. Theselected cells can then be grown to large numbers using large scalefermentation techniques known to those skilled in the art biotechnology.

However, there is a potential problem with removing the short chainalkane metabolism genes. The genes that encode for the metabolism oflight chain hydrocarbons may be in clusters with the genes that arerequired for the production of useful metabolites such as surfactantsfor the emulsification of oil. Because surfactants are secreted to helpthe transfer of short chain alkane across the cell membrane they may becombined with the alkane metabolizing genes or controlled by the samepromoter. In that case the up-regulation of useful metabolites and thedown-regulation of light chain metabolism may require more complex genemanipulation. That is, key enzymes for the metabolism of short chainalkane should be inactivated not the entire gene cluster related toalkane consumption.

One problem that can prevent the success of this approach is that thegenes that code for hydrocarbon emulsification of oil, which helps oilrecovery, add no benefit to the microbe if the oil is not being consumedby the bacteria. Also, if the bacterial culture has a preferred carbonsource in the waterflood fluid, the genes for surfactant productionwould quickly be lost. A microbial population will generally only carrythose genes that are necessary for it to prosper in an environment. Ifthese genes are not needed, they are soon lost. This is why thenutrients, and especially the carbon source, must be carefullycontrolled if the process only depends upon wild type microbes torecover oil from old wells. Therefore, some advantage must be given tothe engineered microbes to make them survive better in the oil reservoirenvironment. The engineered microbes that can only metabolize highmolecular weight oil or produce oil emulsifying surfactants must have acompetitive advantage over indigenous microbes that can metabolize shortchain alkanes.

It is the object of this invention to provide microbes with genes thatare useful for the enhanced recovery of petroleum oil from undergroundreservoirs, oil sands and other sources of heavy oil while suppressingthe consumption of the lighter fraction of the petroleum. In addition,it is the object of this invention to give the host or recipientorganism of these genes a competitive advantage for the specialenvironment of the hydrocarbon resource reservoir. By means known tothose skilled in the art of molecular biology, genes that are isolatedfrom bacteria and Achaea that are indigenous to oil reservoirs ornaturally occurring oil seeps that provide beneficial mechanisms forenhanced oil recovery are engineered into and expressed at high levelsin host microbes. The host microbes are chosen for their survival in theextreme environment of an oil reservoir. The host microbes are providedwith a selective advantage for the reservoir environment. In the presentinvention, and in a specific case, the selective advantage is high pHtolerance. In addition, as discussed earlier, the microorganisms used inthe methods of the present invention are also deficient in their abilityto utilize short chain hydrocarbons but has the ability to converthydrocarbons into fatty acids that can be useful for emulsifying the oilat alkaline pH. In addition, the engineered or modified microbes canhave the ability to utilize a special energy and or carbon source thatis supplied in the waterflood fluid. Genes that code for consumption ofheavy oil or toxic petroleum components are also beneficial to both themicrobe and oil recovery process. These beneficial genes can bepreserved or transferred into the engineered microbes.

Methods designed to stop or reduce the consumption of beneficial lightweight petroleum by the consortium of microbes that is used to makesurfactants and other metabolites that are beneficial to enhanced oilproduction are described in co-pending U.S. application Ser. No.12/869,647, published on Mar. 24, 2011 as U.S. application PublicationNo. 20110067856, the entire disclosure of which is expresslyincorporated by reference herein. U.S. application Publication No.20110067856 also discloses a microorganisms which, in addition to beingdeficient in their ability to degrade short chain hydrocarbons of about12 carbons or less are capable of growing in an environment of highsalinity, and methods and means for their selection and preparation bytechniques of genetic engineering.

According to one embodiment, the consortium used in the methods of thepresent invention will include bacteria which additionally have theability to live and grow in an environment of high salinity.

The technology of the present invention is implemented by inoculating anoil reservoir with a culture of one or more microbes each containingcombinations of genes for the various mechanisms that are beneficial forimproved oil production. The methods of the present invention allow fora wide variety of designs, and thus a combination of mechanisms may bedesigned for a particular type of reservoir. In addition, a means forcontrolling and maintaining high expression of these genes may beprovided. In certain embodiments, along with the microbes, the presentinvention also provides the chemical component to create the rightenvironment for the microbes that also suppresses the indigenousmicrobes that might consume the mobilized oil, especially the shortchain alkanes.

In this example, a high pH requiring culture of microbes, are inoculatedinto an alkaline waterflooding fluid such as sodium bicarbonate, sodiumcarbonate or sodium hydroxide used to recover oil. This process is knownin the petroleum industry as a method of recovering oil and is oftencombined with polymers and surfactants in a process known as alkalinesurfactant polymer flooding (ASP). A more detailed description of thisprocess is reported by Hsu et al. in U.S. Pat. No. 6,022,834 and is hereby incorporated by reference.

Increasing the level of alkalinity in the reservoir is likely to betoxic to the indigenous microbes, but the high pH is preferred for theculture of inoculating engineered or selected microbes. In the preferredmode, the indigenous organisms, which might consume light weight oil orproduce hydrogen sulfide, will be inhibited or killed. Therefore, theadded nutrients will benefit only the growth of the processed-designedmicrobes and not the growth of detrimental indigenous microbes. In thepreferred embodiment the inoculating microbes will grow rapidly at thealkaline pH of the process and produce fatty acids from the oil andother carbon sources that will emulsify the residual oil at alkaline pH.As oil is emulsified and swept away by the drive fluid, new oil surfacesare exposed to the alkaline microbes.

In addition, or as alternatives to, gene manipulation, the control ofmetabolic pathways within microbes can be achieved by the use ofchemical compounds that affect the function of one or more enzymes inthe metabolic pathways. Because it is difficult to manipulate thegenetic make up of the indigenous microbes that are present in an oilreservoir at the time of inoculation with the engineered microorganisms,the metabolic pathways of the indigenous microbes are preferablycontrolled by chemical inhibitors, which are discussed herein below.

(1) Isolation and Selection of Oil Recovery Genes that Code for Proteinsand Pathways for MEOR

Over 100 oil degrading microbes have been isolated and reported. Manyhave been well studied and the sequences of genes related to variousfunctions of the petroleum oil degradation process published. In somecases, for example Alcanivorax borkumensis SK2, the complete genome of3,120,143 base pairs (bp) has been sequenced and published (Schneiker Set al., “Genome sequence of the ubiquitous hydrocarbon-degrading marinebacterium Alcanivorax borkumensis.”, Nat Biotechnol, 2006 August;24(8):997-1004) and is available from the NCBI Genome Project Database(NC_008260; GenBank AM286690). A. borkumensis SK2 is a marine bacteriumthat uses oil hydrocarbon as its exclusive source of carbon and energy.It grows on predominantly alkanes and often becomes the dominant microbethat may comprise 80% of the microbial community in an oil contaminatedenvironment. Bacteria of the Alcanivorax genus belong to a larger groupof hydrocarbonoclastic bacteria that also includes the genera ofCyclolasticus, Marinobacter, Neptunomonas, Oleiphilus, Oleisprira andThalassohtuus. These bacteria are able to metabolize both aliphatic andaromatic hydrocarbons. These bacteria represent a good source of genesthat are involved in hydrocarbon utilization pathways.

With the advent of rapid and inexpensive genome sequencing, thebacteria's genes and their roles in hydrocarbon degradation, surfactantproduction and gene regulation are becoming available. Databases such asGenBank, Swiss Prot, and others provide extensive genomic sequence datafrom these hydrocarbon degrading microbes. This data can be searchedwith computer programs such as BLASTX and BLASTN at the National Centerfor Biotechnology Information. In addition, the use of PCR amplificationbased on probes with complementary sequences from the highly conservedsequences for enzymes, known to be needed for hydrocarbon degradation,can be used to isolate and characterize homologous genes from newmicrobes from oil contaminated sites and oil reservoirs. This can bedone to analyze the change in protein sequence that has evolved to adaptto different environments. Such methods would be a useful way to findenzyme sequence modification that evolved as an adaptation to thespecific environment. For example, microbes isolated from an alkalineand hydrocarbon site could contain enzymes that could degradehydrocarbons at high pH. These enzyme sequences could be compared tohomologous enzymes that function in a neutral environment to understandhow to modify the sequence of proteins from neutral pH microbes to befunctional in alkaliphilic microbes.

In the case of A. borkumensis SK2, various gene clusters have beenidentified that are required for; the degradation of short chainalkanes; the degradation of large alkanes up to 32 carbons in length;and the degradation of branched aliphatic and alkylcycloalkanes. Part ofthis process of hydrocarbon metabolism is the production of surfactantsfor the emulsification of various types of hydrocarbons. In the case ofthe smaller or lower molecular weight chain hydrocarbons, theemulsification aids in the transfer of the hydrocarbon across the cellmembrane so that it can be metabolized. Therefore, this gene clusterincludes genes useful for mobilization of oil which could be transferredto a host microbe. It also contains genes that are not wanted, such asgenes that code for the proteins that are needed for the transfer ofsmall alkanes into the cell for further breakdown and consumption.According to the present invention, the unwanted genes are nottransferred to a host microbe or are inactivated or repressed. Genesrequired for the metabolism of larger alkanes, or alkylcycloalkanes, orpolycyclic aromatic hydrocarbons would be candidates for transfer intohost microbes.

The enzymes that oxidize short chain alkanes all the way to a fatty acidsuch as alcohol dehydrogenase or aldehyde dehydrogenase could be removedor modified. This pathway could be stopped at the alcohol or aldehyde orslowed down to provide short chain alcohols as co-surfactants.Alternatively, genes for the entire pathway of short chain hydrocarbonscould be deleted to prevent the reduction of oil viscosity by removal ofsmall alkanes. In the preferred mode, an obligate alkaliphile orhalo-alkaliphile is engineered to serve as a host. The host alkaliphileis given the genes that are required for degradation of all thepetroleum components that would be beneficial to convert into the acidsand alcohols most useful for reduction of IFT and emulsification ofresidual reservoir oil. To compensate for the slower metabolism of theselarger and more recalcitrant hydrocarbons and the lack of small fattyacid for utilization, the host microbe may also need a soluble carbonand energy source to maintain growth.

Many oil degrading microbes are useful source of genes that code forproteins to make products for the mobilization of petroleum oil. In oneexample, one or more bio-surfactants may be secreted by the cells to aidin the emulsification of the oil droplets so that the oil can beabsorbed through the cell wall. Several strains of Bacillus subtilis andBacillus licheniformis have been used to produce a lipopeptide namedsurfactin at commercial scale. This lipopeptide is also useful as anemulsifier and an antibiotic. Bacteria can be used to produce thesesurfactants in fermentation with manipulation of environmental andnutritional factors to increase yield as described by Cooper et al. in1981, Appl. Environ. Microbiol. 42:408-412, by Javaheri et al. in 1985,Appl. Environ. Microbiol. 50:698-700, and Guerra-Santos et al. in 1986Appl. Microbial. Biotech. 24:443-448. More recently, Mulligan et al.reported in U.S. Pat. No. 5,037,758 a genetically modified strain of B.subtilis ATCC #21332 with a mutation at a site in a gene of wild type B.subtilis that is able to produce surfactin at much higher concentrationsthan the wild type. Therefore, by using gene transfer techniques thesewell studied genes, encoding for surfactant production, can betransferred into various host cells and the production of surfactantcontrolled.

There are many types of bio-surfactants that could be useful in theemulsification of oil. Pseudomonas aeruginosa and other species canproduce rhamnolipids, which have a different structure than surfactin,but still function to immobilize oil. Other surfactants, such assophorolipid and mannosylerythritol lipid and glycolipids are producedby various strains of Candida. Over 200 different variations ofbio-surfactants have been reported. The different surfactant structureshave varying degrees of effectiveness depending on the pH, saltconcentration and other environmental factors.

In addition to surfactants and bio-polymers that are functional at highpH and or high salt concentrations, the fatty acids produced from thefirst few steps in the biodegradation of hydrocarbons found in petroleumwill serve as a surfactant at alkaline pH. Unlike the complexsurfactants such as surfactin and rhamnolipids, moderate size fattyacids in the range of about 13 to 24 carbons can be synthesized directlyfrom hydrocarbons found in petroleum with just a few extracellarenzymes. Various isolation techniques could be used to isolatealkaliphiles or alkaline tolerant microbes from high alkaline and oilcontaminated site or underground aquifers. This could provide bothmicrobes and genetic information that would be useful in selecting andengineering a culture of microbes for high pH alkaline or ASPwaterflooding EOR as a combined oil recovery process.

The process of identifying new genes based on DNA sequence similarity orhomology to known gene sequences of similar function is well known tothose skilled in the art of molecular biology. Several methods have beenused in the past. One method is to make probes of complementary RNAsequence with florescent or radio-labeled tags that will bind to mRNA ofthe genes being expressed by the bacteria in the environment. A secondtechnique is to use PCR amplification of DNA isolated from theenvironment with probes made from conserved sequence regions of thesought after genes. A third method used for screening for bioactivitiesis taught by J. M. Short in U.S. Pat. No. 6,030,779. With any of thesemethods, new gene sequences can be isolated from environments ofinterest such as an oil reservoir that is currently undergoing asuccessful MEOR operation. An alternative is an extreme environmentsimilar to one that might be encountered in an oil reservoir during analkaline waterflood.

Another group of oil degrading microbes that are a good source of genescoding for useful products are microbes that can only metabolize highermolecular weight or complex hydrocarbons. For example, Banerjee et al.in U.S. Pat. No. 5,013,654 (1991) reported strains of an aerobicbacterium that will grow on paraffins of chain length longer than 12carbons. They also isolated a mutant strain of P. aeruginosa SB-1,designated SB-3, which has the property of growing on solid paraffins incrude oil of 20 carbons or more, but will not grow on the liquid lighterchain hydrocarbons. Bacteria such as SB-3, which was deposited in theAmerican Type Culture Collection, Washington D.C. as P. aeruginosaA.T.C.C. 39615 contain the genes for extracellular degradation andmetabolism of heavy petroleum oil. These genes, and others isolated bysimilar means, can be transferred into a host microorganism that is ableto thrive in the extreme environment of an oil well. This ability togrow in a well and degrade heavy oil, combined with the ability toproduce various surfactants and biopolymers, and without the ability toconsume light oil is useful in recovery of light weight petroleum. Ifsuch microorganisms could also use a simple carbon source they couldgrow fast and predominate the micro-flora of a reservoir. In addition,the engineered microbe could be given a toxin resistance gene inaddition to alkaline tolerance as a further competitive advantage overthe indigenous microbes that could consume the light weight oil.

More recently, Lei Wang et al. (PNAS Mar. 27, 2007, vol. 104(13):5602-7) reported the genomic sequence of a thermophilic Geobacillusisolated from a deep oil reservoir that could grow on long chain alkanesup to C36, but was unable to grow on short chain alkanes. Their analysisof the genomic sequence showed that it did not contain any homologousgene sequences to the AlkB genes that code for the membrane boundmonooxygenases that oxidize short chain alkanes. This group alsoreported a soluble and extracellular enzyme for the oxidation of longchain alkanes to the corresponding alcohols. This is an example of agene that can be incorporated into a microbe for the conversion of longchain alkanes into molecules better suited to mobilize the oil into amicelle. To further convert the alcohol into a fatty acid that wouldhave a negative charge at alkaline pH, two additional enzymes would needto be expressed by the engineered cell. The additional enzymes neededare an alcohol dehydrogenase to convert it to an aldehyde and analdehyde dehydrogenase. Genes for these enzymes needed for long chainfatty acid production could be copied from Geobacillus or anothermicroorganism such as Mycobacterium vanbaalenii PYR-1 or Petrotoga sp.AR80.

Microorganisms isolated from heavy oil reservoirs or other oilcontaminated locations are likely to contain genes for all types ofhydrocarbon metabolizing pathways. The membrane bound monooxygenasesevolved in the transport and oxidation of light chain alkanes can bedifferentiated from extracellular enzymes required for the oxidation ofhigher molecular weight hydrocarbons which are too large and insolubleto transport across the cell membrane. If enzymes from these pathwaysare used in the engineered strain, then sequences modification may berequired so that the enzyme are secreted and resides on the other cellmembrane. Secreted enzymes must be functional in the alkaline pH andhigh salt concentration of the drive fluid used in ASP waterflooding.

In addition to degradation of high molecular weight paraffins, microbesmay be able to degrade other unwanted hydrocarbons in petroleum oil.Polycyclic aromatic sulfur containing hydrocarbons such as thiophenesand dibenzothiophenes (DBT) can be present in petroleum at high enoughlevels that they are toxic to bacteria and detrimental to the refiningprocess. The presence of sulfur compounds in oil will reduce the valueof the recovered oil. The sulfur is generally removed prior to refiningby expensive chemical processes. The need for a lower cost process hasencouraged the development of biological processes based on severalspecies of bacteria that have been isolated that can degrade thesesulfur compounds.

In one example, Rhodococcus sp. Strain IGTS8 converts DBT to2-hydroxybiphenyl (HBP) and inorganic sulfur. The pathway requires twomonooxygenases and a desulfinase. In addition to sequencecharacterization, these enzymes have been improved by site directedmutagenesis to broaden the substrate specificity to include thiophenesand benzothiophenes. A more detailed description of the pathway is givenby Gray, K. A. et al. in Nature Biotechnology 14; 1705-1709 (1996).Although this biodesulfurization of crude oil is efficient at removingsulfur with little reduction in fuel value its wide spread use has beeninhibited by the cost of operating large stirred and aerated reactors.The reactor cost problem can be eliminated by transferring the genesthat code for the proteins in the metabolic pathway into a hostalkaliphile in such a way that they function in the higher pH of an oilreservoir undergoing alkaline waterflooding. These proteins couldsimultaneously degrade the sulfur containing hydrocarbons at the sametime as the oil is being emulsified and swept from the reservoir duringalkaline waterflood-MEOR.

An intermediate of the dibenzothiophene DBT desulfurization pathway2-hydroxybiphenyl-2-sulfinate can be used to aid in the solubility ofpetroleum. Four genes in Rhodococcus erythroplis IGTS8 are known to codefor four enzymes that catalyze each step of the desulfurization pathway.This has been used at large scale to remove sulfur from oil. Similargene sequences have been found in more than a dozen different species ofmicrobes including three of the genes found in Oceanobacillus iheyensiswhich is a halotolerant and alkaliphilic deep sea microorganism. O.iheyensis lacks the DszB gene which codes for the last enzyme thatconverts the sulfinate to 2-hydroxybiphenyl and sulfite or the last stepin desulfurization. A pathway could be engineered to either stop at thesulfinate or the 2-hydroxybiphenyl, whichever compound was best atemulsifying the oil droplets at the reservoir's chemical and temperatureconditions. In this mode, the sulfur level of the produced oil isdecreased. In addition, the product of desulfurization could aid in theoil recovery as another emulsifier.

In summary, the preferred microbe of the present invention (i) containsfunctional genes for the extracellular modification of high molecularweight hydrocarbons at alkaline pH; (ii) lacks functional genes for thetransport and oxidation of short chain alkanes at the cell membrane;(iii) contains the genes for the production of useful compounds for oilrecovery and mobilization such as surfactants and polymers that arefunctional at alkaline pH; and (iv) is regulated to express the usefulcompounds at high levels even if given a simple carbon nutrientsupplement. In a preferred embodiment, the microbe is capable offunctioning and growing in the high pH environment of a petroleumreservoir undergoing alkaline waterflooding. Also in the preferredembodiment, the alkaliphilic microorganisms grow more vigorously on theoil at alkaline pH than the indigenous neutralphilic microorganisms. Inanother preferred embodiment, the microorganism is capable offunctioning in either an aerobic or a limited oxygen environment. Withprevention of short chain alkane degradation, the interdiction of aircontaining oxygen is able to speed growth and oxidative degradation oflarge high molecular weight hydrocarbons into smaller light weighthydrocarbons or more water miscible compounds for the reduction of oilviscosity. In addition, the petroleum's content of sulfur and nitrogencan be reduced, if desired.

(2) Selection of Extremophiles

Microorganisms that thrive in environments that would kill mostorganisms are referred to as extremophiles. These environments maycontain organisms from all three domains although generally are almostexclusively populated by prokaryotes, many which belong to the Archaeadomain of organisms. One type of extreme environment is an alkalineenvironment. Naturally occurring aquatics of extremely high pH haveexisted on the earth for many years. Current examples are soda lakes orthe alkaline lakes or alkaline mineral springs, as well as somepetroleum reservoirs, all of which have existed for many years at pH 9.0or higher. This has allowed the evolution of organisms that have adaptedto these consistently alkaline conditions. In addition, manmadecontaminated sites, such as Lake Calumet in Illinois, have become highlyalkaline after about one hundred years of industrial waste dumping. G.S. Roadcap et al. in Ground Water vol. 44, no. 4 pages 511-517, 2006reported actively growing Bacillus and Clostridia in water up to pH13.2. Unlike most natural soda lakes, this high pH waste site was nothigh in salt. A waste site of low salt concentration could provide foralkaliphile that could function at high pH but that would not requirehigh salt concentration as well. Microbes have also developed genescoding for the resistance to these toxic or extreme environments throughan evolutionary process that may have taken many millions of years. Someresearchers believe that these extreme environments are morecharacteristic of the earth when life first began.

These extreme environments can provide sources of both microbes andtheir genetic information that can be transferred into the appropriatehost microbes that are capable of functioning in the extreme environmentof an oil reservoir. In the case where a petroleum reservoir containsindigenous microbes that are detrimental to oil recovery (light oildegraders), the pH of the waterflood fluid is adjusted to an alkalinelevel that is toxic to the indigenous microbes or inhibits the cellmembrane enzymes needed for the up-take and metabolism of short chainalkanes. The fluid is maintained at a pH within the preferred rangefavorable to the engineered strain and within the preferred pH range forthe secreted enzymes that can catalyze the conversion of hydrocarbons toalcohols and fatty acids. This adjustment can be by waterflooding with afluid as part of the oil recovery process. Therefore, the selection ofalkaliphilic microbes for use in oil recovery is the basis of thismethod. This invention provides methods of developing a culture ofmicrobes that will carry out an oil recovery process without theunwanted consumption of short chain alkanes. In prior methods of MEOR,by simply stimulating the indigenous microbes in petroleum reservoirs,the consumption of short chain hydrocarbons could cause reduction in oilviscosity.

High pH and the combination of high pH and high salt environments can beinhabited by alkaliphilic and haloalkaliphilic microbes from bothdomains; bacteria and Archaea. Aquatic environments can be variable inpH or consistently high pH. Alkalitolerant (high pH tolerant) microbescan inhabit both variable and consistently high pH aquatic environments.These are different than true alkaliphilic microbes that inhabit onlyconsistently high pH environments. Alkalitolerant microbes can functionand grow over pH 9.0, but, have a preferred pH near pH 7.0.

Microbes that can live in a high pH environment often have a cytoplasmicpH higher than pH7.0, but still less than the external environmental pH.The cytoplasmic pH can be estimated by determining the pH optimum ofinternal cell enzymes. External enzymes either engineered into thealkaliphilic host microbe or normally found in the wild-type hostalkaliphile should have a pH optimum within the range of pH used foralkaline waterflood. If the enzymes intended to convert the ranges ofhydrocarbons to alcohols and fatty acids does not have a pH optimum highenough for the alkaline waterflood, then the pH optimum of the enzymescan be shifted by changes to amino acids residues on the surface of theenzyme or in the reactive site or binding sites.

Normally the proteins and other molecules that make up microbial cellswill not function at a high pH. In order for enzymes and other compoundswithin the cytoplasm of an alkaliphile to function in the higher pHsolution often found in alkaliphiles, there must be changes made to thesurface of the proteins and to the pH optimum of the enzymes. Proteinscan be altered in their number and type of basic amino acids that are onthe surface of the protein. For example, the basic amino acid lysine isoften replaced with Arginine. Analysis of the genomic sequences of theseobligate alkaliphiles from both archaeal and bacterial examples indicatean increase in some amino acid residues and a decrease in other aminoacid residues. A more detailed review is given by Tsuyoshi Shirai et al.in Protein Engineering vol. 10 no. 6 pp. 627-634, 1997 and KokiHorikoshi, Microbiology and Molecular Biology Reviews, December 1999, p.735-750. And “Alkaliphiles” 1999 ISBN 90-5702-458-6 published by KodanhaLtd Tokyo Japan, author Koki Horikoshi.

Changing one base pair of a three base pair codon is a single mutationand will not result in a charge change of the amino acid it codes for.Changing from a lysine to an aspartic acid requires a change in twobases in the codon (Lys, AAA or AAG to Asp, GAU or GAC). To make achange this radical, from a basic to acidic amino acid, requires adouble or triple mutation of the codon's base pairs. These radicalchanges are the type found in homologous proteins as seen in thecomparison of alkaliphile to non-alkaliphilic microbes. They areunlikely to occur from simple point mutations, which would not result insuch large charge differences. Therefore, this type of adaptation wouldbe slow and not likely to occur in a species for many years.

Adaptation is unlikely to occur as a result of simple horizontal genetransfer from a non-alkaliphilic microbe into an obligate alkaliphile.Genes from non-alkaliphiles or from neutral pH cytoplasm microbes mustfirst be modified so that the enzymes they code for will have high pHoptimums. This key feature of high pH cytoplasm in obligate alkaliphilescan be the basis for a means that prevents the unwanted gene transferfrom most other bacteria, since most bacteria have neutral pHcytoplasms. If the indigenous microbe is not an alkaliphile, the genesfrom other indigenous microbes in the petroleum reservoir will notproduce enzymes with the preferred pH optimum to function in an obligatealkaliphile especially enzymes that are membrane enzymes involved withthe transfer and conversion of short chain alkane and aromatics. If anunderground oil reservoir contained a large population of microbes thatcould metabolize the light weight oil, these unwanted genes could not bepicked up by the engineered obligate alkaliphilic microbe. To befunctional the indigenous genes would have to go though major changes sothat they would be functional in the high pH cytoplasm or membrane ofthe alkaliphilic host microorganism. Generally, oil reservoirs that arenear neutral pH would be unlikely to contain microbes that couldcontribute functional genes to obligate alkaliphiles.

In certain embodiments of the present invention, new genes are added toa host alkaliphilic microbe. After a microbe is selected for use in analkaline waterflood of the reservoir, it may be desirable to add genesfor the degradation and use of high molecular weight hydrocarbons and/orthe production of surfactants and polymers. If these genes aretransferred from other microorganisms, it may be necessary to modify thegenes for high expression and function of the encoded enzymes with ahigh pH optimum. This can be done by a combination of rational proteinsequence design and site directed mutagenesis. Therefore, the proteinsand enzymes required for the production of a surfactant or a hydrocarboncleaving enzyme useful for oil emulsification, can be engineered into analkaliphile after the gene sequences are changed to make the proteinsmore functional at a pH higher than 9.0.

In accordance with the present invention, if needed, the proteins andenzymes required for the production of a surfactant or a hydrocarboncleaving enzyme useful for oil emulsification are engineered into analkaliphile after the gene sequences are changed to make the proteinsmore functional at high pH. The mutated sequences are evaluated forexpression and activity at pH 9.0. In addition, the engineered microbeswill not acquire the light oil consumption genes from the indigenousmicrobes existing in the reservoir, because the enzymes encoded for areoptimal at neutral pH.

Examples of Alkaliphilic Microbes:

Bacillus alcalophilus

Bacillus agaradhaerens

Bacillus cohnii

Bacillus vedderi

Bacillus firmus

Bacillus strain YN-2000

Bacillus halodurans C-125

Bacillus licheniformis PWD-1

Bacillus pseudofirmus OF4

Bacillus strain A30-1 (ATCC53841) A thermophilic and alkaliphilicmicrobe isolated from a hot spring area of Yellowstone National Park,USA. Wang, Y. X. and B. C. Saha (1993) J. Am. Oil Chem. Soc., 70,1135-1138

Bacillus cohnii D-6, FERM P-1592. Produces a detergent and H₂O₂resistant alkaline proteases that is also stable at 60° C. Yoshida andHorikoshi (Japanese patent JP 740710, 1972).

Thermomonospora sp. Isolated by George, S. P., et al. (2001) reporting athermostable xylanase. Bioresource Technol., 78, 221-224.

Oceanobacillus iheyensis HTE831. A halotolerant and alkaliphilicbacterium was isolated on the deep sea Iheya Ridge, Japan at 1050meters. It is a new species not belonging to known genera.

Alkaliphilic Archaeal Halophiles:

Alkaliphilic halophiles can be found in hypersaline soda lakes such asLake Magadi in Kenya, Wadi Natrum lakes in Egypt and soda lakes inChina. These could be engineered to produce bio-surfactants and otherbiological oil recovery compounds that were effective at alkaline pH.Generally, alkaline pH is better for oil emulsification. Increasing thepH of the flood water can be done by adding caustic soda and would havethe added advantage of suppressing the growth of endogenous microbesthat might interfere or have detrimental effects on the quality of theoil produced.

Halothermothrix orenii is an anaerobe isolated from a Tunisian salt lakethat grows in 3.4 M NaCl (20% salt) at 68 deg. C. Ref. Cayol J-L et al1994 Int. J. Syst. Bacteriol. 44: 534-540.

Natronobacterium magadii and N. gregoryi are alkaliphilic halophilesbut, not thermophiles that have been isolated from Lake Magadi in Kenya(ref. Tindall et al 1984 ATCC 43099 and 43098). They have a pH optimumof 9.5 and a salt range of 2.0-5.2 M NaCl.

Natronomonas pharaonic (NC_007426, NC_007427, and NC_007428), is analkaliphilic extreme halophile isolated from a soda lake. This Archaea's2.6 Mbp genome was completely sequenced in 2005.

Halalkalicoccus tibetensis (strain DS12^(T)) Isolated from Lake Zabuye,the Tibetan Plateau, China.

Halalkalicoccus jeotgalt B3

Natronococcus occultus (NCIMB2192^(T))

Thermococcus alcahphilus sp. (DSM10322) a hyperthermophilic archaeumisolated from a marine hydrothermal system at Vulcano Island, Italy. Theoptimum pH is 9.0 at a optimum temperature of 85° C.

In addition to the microorganisms listed above, a larger list isprovided by Enache, M. et al. in the International Journal of Systematicand Evolutionary Microbiology (2007), 57:2289-229, which is expresslyincorporated by reference herein. In addition to the alkaliphiles listedabove, other halophiles can be selected from culture collections orisolated from high pH environments.

(3) Use of Chemical Inhibitors for Control of Metabolic Pathways

The control of metabolic pathways within microbes can be achieved byboth the use of gene manipulation and the use of chemical compounds thataffect the function of any enzyme in the metabolic pathway. Theefficiency of oil recovery will depend on all the microorganisms growingin a reservoir that is undergoing waterflood. This might be acombination of inoculating microbes, selected or engineered microbesthat have the ability to reduce the oil viscosity or emulsify the oilfor higher recovery, but the process might also be affected byindigenous microbes not completely suppressed by the extreme environmentcreated by the waterflood. The inoculating microbes can be designed toalter the crude petroleum is such a way that it more efficiently sweptfrom the reservoir rock. The design process can be by means of genetransfer or inactivation or by the use of chemical inhibitors. Becausethe indigenous microbes have not been selected or modified by genemanipulation they may degrade the crude oil in such a way that itreduces the value and recoverability of the oil. This could counteractor undue the beneficial alterations brought about by the inoculatingmicrobes. Because the indigenous microbes are already present in theunderground reservoir it is difficult to transfer or manipulate theirgenetic makeup. Therefore the use of chemical inhibitors is thepreferred method for control of the metabolic pathways of the indigenousmicrobes.

These chemical inhibitors can be selected or designed to bind to keyenzymes known to be part of degradation pathways typically found in oildegrading microorganisms indigenous to petroleum reservoirs. FIG. 1shows some of the key chemical reactions that are catalyzed by enzymesmade by microorganisms that can utilizes hydrocarbons and the carboxylicacids made from hydrocarbons. In addition to catalyzed chemicalreactions, the transfer of hydrocarbon substrate across cell membranesby transfer proteins may also be blocked by chemical compounds that bindto these transfer proteins. Chemical inhibitors can function by a numberof mechanisms. Some non-limiting examples are molecules that are similarto the substrate and that compete for the reactive binding site with thesubstrate. These inhibitor compounds either cannot react like thesubstrate or if they do react they do not leave the reactive site likethe intended substrate. Other types of inhibitors bind or react with theenzyme at another part of the molecule other than the reactive site andinactivate the enzyme Inhibitors may bind strongly to the enzyme or theymay react chemically with the enzyme to permanently inactivate it. Anyone or a combination of these inhibitors could be used to alter themetabolic pathways of microbes involved in the utilization of crude oil.

In one non-limiting example of reaction or pathway that could beinhibited is the degradation of short chain alkanes. In general thisbiological degradation proceeds by absorption of the alkane by the cellmembrane, conversion to an alcohol by a membrane bound enzyme followedby conversion to an aldehyde by another enzyme and then conversion to acarboxylic acid by another enzyme. If anyone of the steps is slowed downor stopped the detrimental effect of light oil consumption and theincrease in oil viscosity as a result of lighter hydrocarbon loss isprevented.

In some cases the build up and secretion of the intermediates in thepathway could also benefit the recovery of oil. For example, short chainfatty alcohols in the size range of 2 to 8 carbons are useful asco-surfactants in emulsifying oil. In another example, slightly largersize fatty acids in the range of 6 to 20 carbons are useful atemulsifying oil especially at alkaline pH by forming soap molecules thathelp for micelles of oil. In the above examples, a buildup of alcoholcould be induced by the addition of an inhibitor of the enzyme alcoholdehydrogenase of short chain fatty. A buildup of fatty acids could beachieved by inhibiting anyone of the enzymes required for thebeta-oxidation of fatty acids.

A number of chemical compounds have been reported to inhibit thebeta-oxidation of fatty acids. Thijsse G. J. E. in 1964 reported fattyacid accumulation in alkane-oxidizing Pseudomonas (Biochim. Biophys.Acta 84:195-197). In 1979 B. M. Raaka and J. M. Lowenstein reported thatDL-2-bromooctanoate causes complete and irreversible inactivation of3-ketothiolase I a beta-oxidation enzyme (J. of Biological ChemistryVol. 254, No. 14, pp.6755-6762). Salicylic acid was also reported toinhibit 3-ketoacyl-CoA thiolase in P. fluorescens by M. H. Choi et al.in 2008 published online Oct. 3, 2008 DOI 10.1002/bit.22149. Thebeta-oxidation inhibition was comparable to that with acrylic acid andwas believed to be similar but with the added advantage that salicylicacid was not metabolized by the microbe as is acrylic acid and wouldtherefore be longer lasting.

Because of medical importance fatty acids to humans, the inhibition offatty acid transfer across cell membranes has been more studied ineukaryotic cells than in bacteria. At least six of the mammalian fattyacid transfer protein genes have been cloned and their proteinscharacterized (Wu, Q.; Ortegon, A. M.; Tsang, B.; Doege, H.; Feingold,K. R.; Stahl, A. Mol. Cell Biol. 2006, Vol. 26, pp. 3455-3467). Theidentification of small compound inhibitors has been a key technologyfor the development of treatments for obesity, cardiovascular diseaseand fat-induced insulin resistance. High-throughput screening for fattyacid uptake inhibitors in humanized yeast is described by P. N. Blackand C. C. DiRusso in U.S. Pat. No. 7,070,944. Fatty acid transferproteins are also found in bacteria and a report on a family of fattyacid transporters conserved from bacteria to humans is given by Hirsch,D.; Stahl, A.; Lodish, H. F.; Proc. Natl. Acad. Sci. USA, 1998, Vol. 95,pp. 8625-8629.

More recent work has utilized transposon-induced mutants of bacteria ofthe genus Alcanivorax to identify genes required for the export of fattyacid derivatives such as wax esters and polyhydroxyalkanoic acids (PHA).A recent report is given by E. Manilla-Perez et al. was published in J.Bacteriology Vol. 192 No. 3 (2009) pp. 643-656. The export of fattyacids would be beneficial for increasing the acid number of crude oilfor emulsification and the import of short chain hydrocarbons would bebeneficial to inhibit to prevent depletion of light oil from thereservoir petroleum. Isolating the genes that code for each transferprotein would provide a method of screening for inhibitors that do notdecrease export of fatty acids, but that inhibit the import of shortchain alkanes or other light hydrocarbons.

Combination of genetic manipulation and use of chemical inhibitors suchas acrylic acid has been used for production of polyhydroxyalkanoicacids (PHA) because of their use to produce biodegradable thermoplasticsand elastomers. A more detail report of PHA production by recombinant E.coli in combination with the use of acrylic acid inhibitor is given byK. Zhao et al. in FEMS Microbiology letters 218 (2003) pp. 59-64. Morerecent work has focused on the use of microbes that are able to secretethe fatty acid derivatives such as Alcanivorax borkumensis. A recentreport is given by E. Manilla-Perez et al. was published in J.Bacteriology Vol. 192 No. 3 (2009) pp. 643-656.

The effectiveness of any chemical inhibitor can be determined by testinga range of inhibitor concentrations added to a culture of microorganismsgrowing on a sample of crude petroleum or a simple hydrocarbon substratesuch as hexadecane. The secretion of fatty acids into the medium can bedetermined by conversion of the fatty acid to a methyl ester foranalysis by GC. In example 4 a digestion of oil from the Red Riverformation in Montana was used to test a few different chemicalinhibitors of beta-oxidation to measure the inhibitors affect on oil andthe acid content of crude oil.

Further details of the invention are provided in the followingnon-limiting examples.

All references cited throughout this disclosure and the references citedtherein are expressly incorporated by reference herein.

EXAMPLE 1

FIG. 4 shows the alignment of the amino acid sequences of the LadA longchain alkane monooxygenase (SEQ ID NO: 1) with the hypothetical proteinGen ID 9420269 HacjB3_12265 from Halalkalicoccus jeotgali B3alkalitolerant halophiles (SEQ ID NO: 2) and with another hypotheticalprotein from the halophile, Halorubrum lacusprofundi (ATCC 49239) GeneID 7401614 Hlac 0096 (SEQ ID NO: 3). The amino acid compositions for thethree proteins are shown below.

Protein: GI: 134268638_G_thermodenitrificans Length = 440 amino acidsMolecular Weight = 50463.66 Daltons Amino Acid Number Mol % Ala A 286.36 Cys C 5 1.14 Asp D 28 6.36 Glu E 32 7.27 Phe F 18 4.09 Gly G 337.50 His H 20 4.55 Ile I 26 5.91 Lys K 31 7.05 Leu L 33 7.50 Met M 102.27 Asn N 20 4.55 Pro P 16 3.64 Gln Q 10 2.27 Arg R 23 5.23 Ser S 235.23 Thr T 19 4.32 Val V 32 7.27 Trp W 7 1.59 Tyr Y 26 5.91

Protein: GI: 299125497_H_jeotgali Length = 461 amino acids MolecularWeight = 51901.50 Daltons Amino Acid Number Mol % Ala A 44 9.54 Cys C 30.65 Asp D 39 8.46 Glu E 49 10.63 Phe F 21 4.56 Gly G 35 7.59 His H 132.82 Ile I 14 3.04 Lys K 9 1.95 Leu L 30 6.51 Met M 12 2.60 Asn N 102.17 Pro P 21 4.56 Gln Q 15 3.25 Arg R 38 8.24 Ser S 24 5.21 Thr T 286.07 Val V 34 7.38 Trp W 6 1.30 Tyr Y 16 3.47

Protein: GI: 222478535H_lacusprofundi Length = 458 amino acids MolecularWeight = 50806.91 Daltons Amino Acid Number Mol % Ala A 41 8.95 Cys C 30.66 Asp D 47 10.26 Glu E 44 9.61 Phe F 17 3.71 Gly G 32 6.99 His H 153.28 Ile I 10 2.18 Lys K 11 2.40 Leu L 30 6.55 Met M 6 1.31 Asn N 6 1.31Pro P 26 5.68 Gln Q 15 3.28 Arg R 30 6.55 Ser S 27 5.90 Thr T 34 7.42Val V 44 9.61 Trp W 6 1.31 Tyr Y 14 3.06

EXAMPLE 2

Isolation of Microbes from High pH and High Salt Site and Isolation fromHigh pH and Low Salt Sites

Step 1: The Isolation of Microbes from High pH Environments

The isolation of microbes from high pH and high salt sites, or theisolation from high pH and low salt sites is a preferred method ofobtaining host microorganism for an alkaline oil recovery process. Sitesthat have been contaminated with petroleum oil for a long time are mostpreferred because they may also contain microbes that can grow on avariety of hydrocarbons. Some oil wells contain water that is of high pHand has been at high pH for many years. Microbes from alkaline petroleumsites are also likely to be resistant to the toxic effects of petroleumand could be used for oil recovery or as host microorganisms forengineering metabolic pathways useful for oil recovery. Themicroorganisms isolated from these types of sites are also useful as asource of protein sequences of enzymes that are optimized for high pHfunction.

Alkaline environments of consistently high pH that are also high insodium ion concentrations (salinities exceeding 100,000 ppm totaldissolved solids) are the most common. Soda lakes and soda deserts arestable and naturally occurring environments found worldwide. These arethe best for isolation of alkaliphilic and halophilic microorganisms.Environments that have variable pH and salt concentrations are morelikely to contain alkali-tolerant and halo-tolerant microorganisms.Alkaline sites containing liquid hydrocarbons, such as petroleum oilfields or waste oil/brine disposal pits or evaporation ponds are goodsites for isolation of microbes that are alkaliphiles orhalo-alkaliphiles and also have the ability to metabolize various typesof hydrocarbons. Groundwater high in calcium ions at high pH is lesscommon, but is a good source of alkaliphiles that are not alsohalophiles and can grow in low concentrations of salinity.

Microbes selected for use in MEOR may be required to function in the lowoxygen environment of an oil well. Facultative anaerobes are ideal hostmicroorganisms because they can survive exposure to oxygen. Especiallygood are microbes that can use nitrate as an election acceptor. Aerobicmicrobes may be used in applications where large amounts of air can beinjected with the waterflood fluid. Microbes isolated from theseenvironments can also be a source of genes or gene sequence informationthat can be used to genetically modify a microbe or engineer a microbeto be tested and used on oil reservoirs of high pH or where an alkalinebrine is used as waterflood.

In one example, H. Al-Awadhi, et al., in Appl. Microbiol. Biotechnol.2007, 77:183-186, reported the isolation of the strains; Marinobacter,Micrococcus, Dietzia, Bacillus, Oceanobacillus, and Citricoccus thatwere able to grow on a range of alkanes and aromatic compounds as solecarbon and energy sources. These microbes were isolated from theintertidal zone of the Arabian Gulf coast. Petroleum oil pollution isacute in an oil-producing area such as the Arabian Gulf. The longcoastline of the Gulf comprises areas in which tidal water is trappedand becomes saline and alkaline as a result of evaporation. Thereforesites containing both oil and high salinity at alkaline pH are likely tocontain alkaline microbes that can utilize various hydrocarbons.

Following the method used by Al-Awadhi et al., microbes in biofilmscoating gravel particles are scraped off with a sterile toothbrush intosterile water. A count of alkaliphilic oil-utilizing microbes is made bygrowth on an inorganic agar medium described by Horikoshi 1998,Alkaliphiles. In: Horikoshi K, Grant W D (eds) Extremophiles: microbiallife in the extreme environments. Wiley-Liss, London, pp 155-179. Thehalophiles were counted on inorganic medium described by Sorkhoh et al.(1990). Representative colonies were isolated and purified on the abovesolid media. The pure cultures were identified by analyzing theirgenomic deoxyribonucleic acid (DNA) sequences. Genomic DNA was extractedby a DNA extraction kit (Sigma, USA). The 16S ribosomal ribonucleic acid(rRNA) was amplified by polymerase chain reaction (PCR). The 550-bpfragment of the 16S ribosomal DNA was enzymatically amplified with theuniversal eubacterial primer combination GM % F with the sequence5′-CCTACGGGAGGCAGCAG-3′ (SEQ ID NO: 4) and DS907R with the sequence5′-CCCCGTCAATTCMTTTGAGTTT-3′ (SEQ ID NO: 5) (Santegoeds et al. 1998Appl. Environ Microbiol 64:3731-3739). The PCR products were purifiedand sequenced. Sequences obtained were compared to those of knownbacteria via the GenBank database and deposited under the accessionnumber DQ646492-DQ646515.

The isolates were surveyed for growth on medium containing 0.5% ofindividual hydrocarbons alkanes from C-13 to C-40 and the aromaticcompounds naphthalene and phenanthrene as sole carbon sources. The pH ofthe medium for the alkaliphiles was maintained at pH 11. It was foundthat the optimum pH for growth of oil-utilizing alkaliphilic isolateswas between pH 8 and pH 10. All isolates could tolerate alkalinity up topH 11 but not pH 12.

Al-Awadhi et al. found that most of the isolates could utilize a widerange of alkanes from C-13 to C-40 and the two aromatic hydrocarbonstested. However, a few could only utilize a narrow range ofhydrocarbons. This method could be used to isolate alkaliphilic wildtype microbes that only had the ability to degrade high molecular weighthydrocarbons. By these methods, pure strains could be obtained ofhalophilic and alkaliphilic microbes that could degrade only certaintarget ranges or types of hydrocarbons. Alternatively these isolatestrains could be sequenced to find variations in enzymes that are knownto degrade certain size ranges of hydrocarbons, such as themonooxygenase LadA, which is an enzyme that converts alkanes longer than15 carbons to alcohols, or NidA3B3 that is a degrading enzyme ofpolycyclic aromatic hydrocarbons. The sequence information could be usedfor adapting other enzymes for function at high pH or salinity byhomologous amino acid replacement.

Less is known about the microorganisms found in environments that arelow in salinity, but high in alkalinity and calcium. The microbialcommunities may not develop as well because of the lower concentrationof nitrogen and phosphorous. Industrial waste water ponds, such as sitesof cement and indigo production, are good sites to explore for low saltalkaliphiles. G. S. Roadcap et al. Ground Water 44, No. 4:511-517 (2006)reported finding more than 100 strains of Bacillus and Clostridium bymicrobial 16S ribosomal RNA sequencing from a high pH and low sodiumground water site at Lake Calumet, near Chicago Ill. The pH of the waterfrom this site is over 13. The Lake Calumet site is also known to havehydrocarbon contamination and is likely to contain hydrocarbon degradingmicrobes that are also able to grow at high pH. By using a proceduresimilar to Al-Awadhi et al. with a medium low in sodium, strains ofmicroorganisms could be isolated from a site such as the Lake Calumetsite. Those microbial isolates could be tested for the ability toutilize either light or heavy hydrocarbons.

Step 2: Isolation of DNA and Genes Needed for Surfactant Production andLiquid Oil Consumption

Microbial strains selected for high and effective surfactant productioncan be further characterized by gene sequencing. DNA is extracted frompoly-carbonate filters as described by Minz et al. (1999) Appl. Environ.Microbiol. 65: 4666-4671. This procedure was modified by Kebbouche-Ganaet al. The DNA was electrophoresed, excised from the gel and purifiedwith a gel extraction kit (Genomic DNA purification system PROM, EGA).Purified DNA from selected strains are amplified with specific 16s rRNAarchaeal primers (5′-TTCCGGTTGATCCYGCCGGA-3′(SEQ ID NO: 6) and 5′YCCGGCGTTGAMTCCAATT-3′ (SEQ ID NO: 7)). 16s rRNA sequence informationcan be aligned with rRNA sequence from known alkaliphiles for genera andfamily identification.

DNA or mRNA probes can be based on known genes from an organism thatproduces a surfactant. One example is Pseudomonas aeruginosa, whichproduces rhamnolipid. The synthesis of this glycolipid is by sequentialglycosyl transfer reactions. The genes involved in rhamnolipidbiosynthesis are encoded on a plasmid, and their expression is regulatedby a quorum sensing system. A more complete review is given in Lang andWullbrandt (1999) Appl. Microbiol. Biotechnol. 51:22-32. Other species,such as Bacillus subtilis, produce surfactin, a lipopeptide whichcontains about 7 amino acid residues. Other microorganisms secretehigher molecular weight biosurfactants consisting of polysaccharides,lipoproteins, and lipopolysaccharides. Isolation and identification ofthe surfactants secreted by the isolated strains can be done by HPLCwith a mass-spec detection system. Identification of the chemical natureof the surfactants produced by each isolated strain can be usefulinformation for finding genes that are required for the surfactantproduction and secretion. For example, a glycolipid similar torhamnolipid would likely be dependent on genes similar to those involvedin its biosynthesis in P. aeruginosa. These genes from wellcharacterized microbes could be used to construct probes for findingsimilar genes in the alkaliphilic isolates.

However, if the surfactants produced by the alkaliphilic microbes aretotally new and unlike any other well-studied surface active compounds,then other methods of gene isolation can be used. For example,correlating higher levels of specific mRNAs with production of highlevels of surfactant can be used to find needed genes. If the presenceof alkanes induces the production of surfactant, then the level of mRNAneeded for surfactant production will be increased. The use of DNAmicroarrays can identify the increase in gene transcription to mRNA whensurfactant production is induced, as can whole transcriptome shotgunsequencing of RNA or cDNA (WTSS or RNA-Seq). Also, sequencing of thespecific cDNA, made from the increased mRNA can be used to identify therequired genes sequences.

Based on this method, the identification of genes required for theproduction of surfactant and the degradation of liquid oil can be doneby mRNA differential display. This method was used to identifyCyclohexonone metabolism related genes (Brzostowicz et al. (2000) J.bacterial. 182: 4241-4248). These mRNA techniques make it possible toaccess regulated genes directly without purification of gene products.These approaches are based on comparisons of two cultures and theidentification of genes whose mRNA is more abundant when a metabolicpathway is induced. In the above example, if surfactant production isinduced by the presence of oil, then mRNA that codes for surfactantproduction as well as enzymes for oil metabolism will be at higherlevels compared to the uninduced culture. These techniques rely on thehybridization of DNA on membranes as described by Chuang and Blattner1993 J. bacteriol. 175: 5242-5252. It was by this method thatBrzostowiez et al. that led to the discovery of the genes for twomonooxygenase enzymes responsible for the oxidation of cyclohexanone.This same technique can be used for the identification of genes codingfor proteins and gene products of alkaliphilic microbes that lack enoughsequence homology to bind to probes constructed based on proteinsequence mesophilic homologous enzymes.

Probes may also be based on protein sequence of homologous enzymes withhighly conserved catalytic site and binding sites. In this case a shortdegenerate DNA probe is constructed to bind with any DNA that had thesequence of base pairs that would code for the highly conserved aminoacid residue sequence.

Although all these methods can be successful at isolating new genesrequired for surfactant production and liquid hydrocarbon oildegradation in halophiles, genome sequencing of larger numbers ofobligate halophiles will enable faster identification and isolation ofnew genes.

Step 3: The Prevention and Modification of Short Chain Alkane Metabolism

The expression of genes required for the production and secretion ofsurfactants and for the degradation of high molecular weighthydrocarbons are beneficial to the mobilization of oil. Degradation ofshort chain alkanes and other low viscosity petroleum components is verydetrimental to oil recovery. Therefore, if the genes of a microbe couldbe modified so that the microbe does not metabolize light oil, theviscosity would decrease and the recovery of petroleum would increase.However, this must be done in such a way that production of surfactant,which may be under the control of a single gene promoter, is not alsoprevented. With the loss of liquid oil metabolism, another utilizablecarbon source is needed to offset the loss of energy from the lightchain hydrocarbon metabolism. Often the genes needed for both liquidhydrocarbon consumption and surfactant production are clusteredtogether. Therefore, deactivating or removing the genes needed for shortchain alkane uptake must be done in such a way that the genes needed forhigh production of surfactant are not deactivated or down regulated.

One method of achieving this specific gene modification is by homologousgene replacement. A wild type gene is replaced with a new gene that hasa modified nucleotide sequence and that codes for a protein with adifferent amino acid sequence. This process can be used to make smallchanges to enzymes to change the catalytic efficiency or specificity ofthe enzyme. A change of one or two amino acid residues can disable theability of the new enzyme to bind the same substrate or to catalyze keysteps in the conversion of substrate to product at the same rate. Thisprocess has been used in many genetic systems wherein similar genes arereplaced by a mutated gene by homologous recombination (MolecularBiotechnology edited by Glick and Pasternak, 2003, Chapter 8). Alongwith the mutated gene, a selectable maker is also incorporated so thatnew microbes that have taken up the mutated gene can be selected. Thisprocess requires a certain level of genetic manipulation tools.Fortunately, a gene knockout system has been developed for thehalophilic Archaea, Haloferax volcanii and Halobacterium salinarum basedon the pyrE gene reported by Bitin-Banin et al. in J. Bacteriol. 2003,185: 772-778. This system has been further developed, and now fourdifferent selection principles are available (Allers et al. Appl.Environ. Microbiol. 2004, 70: 943-953) for Hf. volcanii. By using thistechnique or similar gene replacement techniques with selectable makers,the monooxygenase genes isolated from the wild type halophiles can bereplaced with modified gene sequences.

By this process or other genetic manipulation processes, a number ofchanges can be made in the amino acid sequences of enzymes thatfacilitate the uptake or metabolism of light chain alkanes. This processcan be done by random changes to any amino acid in the enzymes sequence,by trial and error. The resultant enzymes with the amino acid changescan be tested for changes in substrate binding, substrate specificity,and rate of conversion to product. In general, most of the randomchanges will have little effect, or will decrease the catalytic rate.This process is much easier if the three dimensional structure of theenzyme is known or can be determined by X-ray crystallographic analysis.In this example the structure of some alkane specific monooxygenaseshave been determined and would be useful in predicting key amino acidsto change. For example, by making point mutations of the amino acidresidues at the binding sites also known as histidine boxes, it wouldlikely prevent or cause a reduction in the rate of alkane metabolism.

Changing any of the amino acid residues, especially the histidines, willaffect the ability of these enzymes to metabolize liquid hydrocarbons. Anumber of these modified enzymes can be evaluated in a modelalkaliphilic host, such as Oceanobacillus iheyensis, to determine theenzymes ability to function at high pH. Modified wild type alkaliphileswith the mutated alkane conversion enzymes can be evaluated at thelaboratory scale for their ability to produce surfactant, but withlimited ability to grow on octane or diesel as a carbon source. From thegroup of engineered microbes, the strains that achieve high levels ofgrowth utilizing an inexpensive carbon source, and that produce highlevels of surfactant, and that consume the smallest amount of lightmolecular weight oil (C6-C8) are selected. The consumption of shortchain alkane can be determined by analysis of remaining oil in thereaction vessel. A more sensitive method is with a carbon-14 isotopelabeled alkane. Small amounts of uptake of the isotopic carbon can bemeasured in the cells. Alternatively, the rate can be determined fromthe isotopic carbon dioxide produced.

From this group of engineered microbes the selected halophiles aretested for their ability to mobilize oil in a laboratory scalewaterflood core sample test. This test consists of saturating areservoir rock core sample or a packed sand column with petroleum oil. Aflow of water or brine is then pumped through the core sample until thefree oil is washed out. Then the microbe culture in a growth buffer isintroduced into the core sample that still contains the residual oil.The core sample inoculated with microbes is left to incubate for one totwo weeks. After incubation, a flow of waterflood buffer is passedthrough the core and the amount of oil removed by this flow is measuredas a function of buffer flow volume. With this small scale laboratorytest, the effectiveness of each of the engineered and wild type culturescan be measured and compared. The improved cultures should show anincrease in the rate and total amount of oil removed from the core.There should also be an increase in the number of microbes, indicatinggrowth in the high salt environment. However, this short test does notindicate oil or light chain alkane consumption because the time that themicrobes are in contact with the petroleum is too short and there is noeasy way to measure the total remaining oil.

Therefore, another test is needed to determine the short chain alkaneconsumption. The conditions of the digestion should match the waterflooddrive buffer or fluid. It should contain the soluble carbon source, suchas molasses, that will be used to supplement growth. However, thesoluble carbon source should not be a catabolite that will causerepression of alkane degradation pathway genes. A report of carbonsources that can cause repression of alkane degradation pathways inPseudomonas putida is given by F. Rojo et al. in the J. Bacteriology2003 185: 4772-4778. The incubation should be long enough (severalweeks) to measure degradation and loss of alkanes or a change in totalalkane hydrocarbon composition or a change to the relative amount ofvarious hydrocarbons if a mixture or sample of petroleum oil is used.The measure of an engineered or selected microbe that will be a goodcommercial candidate is that there is no decrease, or relative smalldecrease, in the lighter weight hydrocarbon. As a comparison, this sametest is performed with the indigenous microbes isolated from thelocation or oil reservoir. A test using only the stimulation ofindigenous microbes might produce less oil, or produce oil with a largerhigh molecular weight fraction. The best cultures will be the ones thatcan produce the most surfactant and the most oil without decreasing thepercentage of light weight oil in the petroleum samples.

EXAMPLE 3

Addition of Fatty Acids of Various Length Alkanes to Crude Petroleum Oilto Experimentally determine the Emulsification of Oil at Alkaline pH.

This test was done by mixing small amounts of carboxylic acids with apetroleum sample. The petroleum sample used was a sour oil from theSleep formation of Pennsylvanian sandstone in Byron, Wyo. The carboxylicacids used in this test were; a 16 carbon (hexadecanoic) acid C₁₆H₃₂O₂,an 8 carbon (octanoic) acid C₈H₁₆O₂, a 4 carbon (butanoic) acid C₄H₈O₂,a three carbon acid C₃H₆O₂ and a 2 carbon acid C₂H₄O₂. The amount ofacid added was approximately enough to neutralize 1.0 mg of KOH per gramof oil or an acid number of about 1.0. The organic acid and oil wereheated to about 50° C. for 30 minutes to dissolve the acid in the oil.The oil was then mixed with the sand at the ratio 3 ml (2.67 g) oil with10 g sand. The sand was 30 mesh play sand from Home Depot. The oil andsand mix were packed into a small column and then eluted with the testdrive fluid by gravity flow. All test elutions were done at a roomtemperature of about 25° C. The elution volume was 20 ml. the oil andaqueous fluid were separated and the dried oil was weighted. The firstelution was with a 1% NaCl brine solution at neutral pH. The secondelution was with a 0.05% NaOH, 1% NaCl brine solution at about pH12. Andthe third elution was a 0.05% NaOH, 1% NaCl, 1% Tween 20 brine at pH 12.The first elution was to test a waterflood with brine. The second was totest an alkaline waterflood and the third elution was to represent analkaline waterflood with chemical surfactant. The results indicate thatthe additional oil elution at alkaline pH was affected by the chainlength of the fatty acid. The longer fatty acids have increased theviscosity of the oil and decreased the elution from sand, which had moreimpact than the soap of the fatty acid help the emulsification of theoil. The shorter chain length fatty acid may have been less soluble inthe oil and more soluble in the water and not been as effective atemulsifying the oil with the brine. This effect is temperature dependantand the elution experiment should be done at approximately the sametemperature as is expected within the reservoir formation. This is alsodone to better determine the preferred size range for the microbiallyproduced fatty acids from the degradation of the reservoir crude oil.

A similar small scale test can be done with the partially bio-degradedcrude oil. The modified oil can be extracted with a standardconcentration of sodium hydroxide and then back titrated to determinethe amount of extractable fatty acids. The extracted fatty acids can bedetermined by conversion to methyl esters for GC analysis. In addition,sand pack columns can be used to evaluate the alkaline elution of thepartly degraded oil. The degree of biodegradation of the oil can beevaluated by a combination of acid number, fatty acid type, GC analysisof degraded and undegraded oil and oil recovery from sand pack columns.The preferred microbes are the cultures that can increase the acidnumber and the type of acids that are best for higher oil elution underalkaline flooding and that do the least degradation of the shorter chainhydrocarbons.

TABLE 1 Results for sand pack column elution Recovered oil (gram) from10 g sand/3 mL (2.67 g) Byron column Con- 1.25 1.875 2.5 5 10 trol mg C2mg C3 mg C4 mg C8 mg C16 1% NaCl 1.14 1.12 1.07 1.16 1.10 1.05 1%NaCl/0.05% 0.24 0.09 0.20 0.33 0.46 0.17 NaOH 1% NaCl/0.05% 0.17 0.140.28 0.37 0.23 0.02 NaOH/1% Tween Total recovered 1.55 1.35 1.55 1.861.79 1.24 oil (g)

EXAMPLE 4

Deletion of Genes in the Alkane and Fatty Acid Degradation Pathway ofHaloferax volcanii

The acyl coenzyme A dehydrogenase protein, encoded by the fadE gene, hasbeen shown in E. coli to be necessary for efficient degradation of fattyacids via the beta-oxidation pathway. In the alkane degrading strainGeobacillus thermoleovorans B23, the fatty aldehyde dehydrogenaseprotein, AldH, was identified and shown to convert various fattyaldehydes to fatty acids. Similarly, a fatty aldehyde dehydrogenaseencoded by ald1 in Acinetobacter sp. strain M-1 was shown to convertvarious fatty aldehydes to fatty acids.

Searches of FadE, Aldh, and Ald1 were carried out in the Genbankdatabase, using BLASTP, to find matches in the genome sequence ofHaloferax volcanii DS2. The best match for FadE consisted of Acd3(YP_003535250.1). The best match for both AldH and Ald1 was AldY5(YP_003533953.1).

Directed gene knockouts were made in the acd3 and aldY5 genes byhomologous recombination in strain GFF127, an isolate of Haloferaxvolcanii DS2 containing a knockout of the pyrE2 gene with a mevinolinresistance marker (i.e., pyrE2Δ::MEV). For gene knockouts, linear DNAsegments were constructed containing 1000 nucleotides of sequenceimmediately upstream of the target gene, followed by the Haloferaxvolcanii pyrE2 gene, followed by 1000 nucleotides of sequenceimmediately downstream of the target gene. The DNA segments weregenerated by a fusion PCR strategy, whereby the three parts of a segment(target gene flanks and pyrE2 gene) were PCR amplified in a first step,separately, from the Haloferax volcanii genome. PCR primers foramplification of the pyrE2 gene at the upstream and downstream endscontained sequences at the 5′ ends that matched the upstream anddownstream flanking sequences of the gene targeted for knockout. In asubsequent step, the three parts of the segment were assembled in a PCRreaction that contained the three parts and PCR primers at either end ofthe entire segment. DNA segments were then transformed into GFF127 byPEG transformation (Dyall-Smith, The Halohandbook: protocols forhaloarchaeal genetics, 2009) and the pyrE2 gene was selected on mediumlacking uracil (Hv-Ca medium). Transformant colonies were streaked forisolation. Replacement of the target genes was confirmed by PCRamplification across junctions at both ends at sites of integration,using a primer internal to the pyrE2 marker and a primer external to theintegration construct, approximately 1.2 kb upstream or downstream. Theapproximately 1.3 kilobase PCR band generated from the genomes ofmultiple mutant isolates, but not from that of wildtype Haloferaxvolcanii, demonstrated successful acd3 and aldY5 gene knock outs (see,FIG. 5). Absence of the target gene in the genomes of mutant isolateswas confirmed by the absence of a PCR product generated using a pair ofDNA primers internal to the gene (491 or 480 base pairs for the acd3 andaldY5 genes, respectively); as a control, the internal PCR product wasobserved when the PCR was carried out in parallel with a wildtypestrain.

EXAMPLE 5

Determination of the Effect of Chemical Inhibitors for Increasing theAcid Content of Oil

Experimental Procedure:

An oil degrading microorganism was used to evaluate the use of chemicalinhibitors to slow the utilization of hydrocarbons in petroleum. In anon-limiting example, three different chemical compounds that have beenreported to inhibit beta-oxidation were tested in oil and culture mediumon a petroleum sample with a strain of Rhodococcus rhodochrous ATCC#53968 in m9 minimal medium. To test the ability of each chemicalinhibitor to slow the consumption of fatty acids a culture of bacteriawas grown on a crude oil sample for a period of three days with crudepetroleum oil as the only carbon source. Four separate flasks were setup, each with an oil to aqueous media ratio of 1 to 5 (10 ml of crudeoil to 50 ml of culture medium). Each of the three chemical inhibitorswas added to a separate flask and a fourth flask was allowed to digestwithout the addition of a chemical inhibitor as a control. The flaskswere shaken at a room temperature of about 25° C. under aerobicconditions. The progress of the microbial digestion was monitored by theoptical density at 600 nm and by visual inspection of the brown oilcolor in the aqueous phase. Three days after the addition of inhibitors,or a total of six days of incubation with oil, a small sample of oil(0.31 g) was removed. A second sample of the same size was remover afteranother 6 days. The organic acids were extracted from each oil samplewith 1% NaOH in 70% ethanol. The samples were shaken vigorously for oneminute. The aqueous extract material was then extracted with hexane toremove any trace of oil. The samples of extracted organic acids werethen dried under vacuum and converted to methyl esters with BF₃ inmethanol.

The mixtures of methyl esters were analyzed by GC (gas chromatography)using a HP (Hewlett Packard) 5890 GC with an Agilent Technologiescapillary column, HP-5 (crosslinked 5% PH) ME Siloxane 0.25 micrometercoating with a length of 30 meter and a column ID (internal diameter) of0.32 mm. Helium was used as a carrier gas. Detection was by a FIDdetector and integration of peak areas was done with a HP 3396integrator. The temperature program held an initial temperature of 40°C. for 1.0 minutes then increased at 15 degrees per minute to 280° C.,and finally held at a temperature of 280° C. for 5 minutes. To quantifythe amount of organic acids extracted by this procedure, a stocksolution of 2-bromooctanoate and 2-bromohexadecanoate were added to asample of undigested Red River crude petroleum oil and then extracted bythe same procedure as the bio-digested oil samples. The average area fortheses two organic acids was used to estimate the amount of organicacids in micromoles per area unit determined by the GC integrator. Thenumber was calculated to be approximately 1.0 micromoles per 100 areaunits. The area units for each of the GC peaks are list in Table 2. Thisnumber was used to estimate the micromoles organic acids extracted fromthe oil. This estimate was used to calculate the number of mg of KOHthat equaled the number of micromoles of organic acid estimated. Thisnumber is listed on the last row in Table 2 as the calculated AN (acidnumber) for each digestion experiment. These estimated numbers are onlybased on organic acids that are hydrophobic enough to stay in the oilphase and not move into the aqueous phase. Organic acids measuredinclude the carboxylic acids made by the bacteria and that have beensecreted out of the cell and have migrated back to the oil phase. Theyare also hydrocarbons that are small enough to be extracted into theethanol aqueous extraction solvent. This number is likely to be lessthan the total acid number (TAN), a measure often used to evaluate crudepetroleum for amenability to alkaline waterflooding.

At the end of the experiment the oil and water were separated and thevolume determined. The volume of oil was 11.5 ml for sample withoutinhibitor, 10.0 ml for the sample with 2-bromooctanoate and 9.0 ml forthe sample 2-bromohexadecanoate. The volume of oil for the sample withacrylic acid had increased to 33.5 ml. This indicated that the oil hadformed a stable water in oil emulsion at some point during theexperiment. This could have lead to a decrease in the amount of oilremoved in the 0.31 g removed at day 6 and day 12 for GC analysis. Thiswould have under reported the amount of methyl esters determined by GCanalysis. The amount of aqueous phase was determined to be 22.5 ml forthe acrylic acid sample and 46 ml, 48. ml and 47.5 ml for the others.This was further evidence that a water in oil emulsion had formed. Atthe end of the experiment the larger volume of one ml of oil was used todetermine the acid number by the alcoholic alkaline extraction of acidsand back titration with acid to neutral pH. This was similar to themethod used for GC methyl ester formation reported by A. G. Shepherd etal. in Energy Fuels 2010, 24, pp 2300-2311 DOI: 10.1021/ef900949m. Theincorporation of aqueous media in the stable emulsion form from theacrylic acid inhibited digestion interfered with the back titration todetermine extracted carboxylic acids. The acid number of the emulsionwas about twice that of the other oil samples. This was withoutcorrecting for the lower concentration of oil in the 1 g of emulsionthat was used. The final analysis acid numbers were 10 mg KOH per gramof oil emulsion produced from the acrylic acid inhibited digestion. Thecalculated acid number for the uninhibited digestion was 5 mg of KOH pergram. The other two inhibited digestions were analyzed to be 4.5 mg forthe 2-bromooctanoic acid and 6.5 for the 2-bromohexadecanoic acidinhibited digestion. The undigested oil was determined to have an acidnumber of 6.5 mg of KOH by analysis of a one gram sample and a 10 gramsample. Ideally this acid analysis should be done on oil samples of 10to 50 grams for best accuracy.

Results:

The integator area units of about 20 GC peaks that were seen in each ofthe digested samples of the Red River petroleum oil are listed in Table2. The undigested oil sample showed a small level of methyl ester peaks.The earlier eluting peaks are believed to represent methyl ester oflighter hydrocarbon carboxylic acids and the later eluting peaks arebelieved to represent methyl ester of the heavier organic acids. As acomparison the peak area units are subtotaled for the peaks elutingbefore 13 minutes compared to the subtotal of the later peaks elutingafter 13 minutes. The total of all selected peaks and a total of all thepeaks integrated by the GC integrator are listed below each column. Thetotal area units for all the peaks integrated by the GC integrator arelisted for the oil samples from Red River oil field in Montana.

Analysis of Results:

The GC analysis of the digestion with the 2-bromohexadecanoate showed avery large peak at 14 minutes, which corresponded to the retention timeof the inhibitor compound. There were also some large earlier peaksbetween 9 and 13 minutes, which made determination of the increase infatty acids from oil digestion difficult. The quantity of this peak wasalso higher than the concentration of 2-bromodexadecanoate added as aninhibitor to the aqueous media. It was assumed that the long chainhydrocarbon inhibitor was absorbed into the oil phase at a higherconcentration. Therefore, the total peak areas reported for thisinhibitor are higher due to the 2-bromohexadecanoate methyl ester. Amuch smaller peak was seen for the 2-bromooctanoate and did not havemuch effect on total peak area. No peak was seen for the methyl ester ofacrylic acid and it is believed to not be retained at 40° C. on the GCcolumn.

Digestion with the bacteria increases the total amount of carboxylicacids in all the samples. The digestions with the chemicalbeta-oxidation inhibitors produce about the same or a little more totalmethyl ester peaks than the bacterial digestion without an inhibitor. Acomparison of the subtotal peak area increases for the digestion withoutinhibitor to the acrylic acid and 2-bromooctanoate inhibited digestionsshows a relative difference in the rate of increase. The later elutingpeaks increase at a faster rate with the inhibitors than without. Forexample, the 12 day sample with no inhibitor increase from 5.9 to 45.9area units for peaks up to 13 minutes. The sample with the acrylic acidinhibitor has an increase of from 5.9 to 43.7 area units, which is lessthan without inhibitor. The later eluting peaks increase from 3.6 to14.3 without the inhibitor, but with acrylic acid the later peak areasincrease to 22.5 area units. This suggests that the inhibitors areslowing down the conversion of large fatty acids to smaller fatty acids.The optical density measurements and the visual inspection of thesamples suggested that the bacteria were growing faster and digestingthe oil faster in the uninhibited digestions. After 6 days of digestionthe optical density had reached 2.23 for the uninhibited digestion ascompared to 0.49 for the acrylic acid inhibited sample and 1.84 for the2-bromooctanoic acid and 2.4 for the 2-bromohexadeconoic acid. After 12days of digestion the uninhibited sample had reached 4.0 and the acrylicacid had increased to 1.7 and 2.2 and 2.4 for the other two inhibitors.Therefore, by this experimental analysis one can determine whichinhibitors increase the carboxylic acid content or the acid number ofthe oil fastest with the least amount of oil consumption. The oilconsumption can be determined by quantitation of remaining oil or bygrowth of bacteria were oil is the only carbon source. It appears thatall the inhibitors helped reduce the utilization of fatty acids and theconsumption of oil while producing an increase in acid number. It alsoappears that acrylic acid was the most useful because it also helpedform a stable water in oil emulsion, which could also improve oilrecovery.

TABLE 2 Digestion of oil with and without inhibitors Red River No AA BrC8 Br C16 No AA Br C8 Br C16 Retention No Inhibitor Inhibitor InhibitorInhibitor Inhibitor Inhibitor Inhibitor Inhibitor Times Digestion 6 days6 days 6 days 6 days 12 days 12 days 12 days 12 days 4.2 0.727 0.58 0.470.354 4.5 0.204 0.242 0.351 0.226 1.512 1.051 0.943 0.237 4.6 0.3741.519 5.6 1.459 0.948 0.835 1.099 3.531 2.754 1.656 0.624 6.6 0.2575.844 2.856 2.783 3.049 9.689 7.835 4.625 1.498 7.6 0.251 5.304 2.7242.476 2.553 7.075 5.761 3.349 1.246 8.2 0.257 0.495 0.703 0.682 0.7050.644 0.558 0.497 0.602 8.5 0.294 1.834 1.907 1.443 1.395 2.141 3.3691.763 0.624 9.4 0.418 0.725 1.575 1.146 1.131 0.531 0.747 0.681 0.47610.2 0.277 0.631 1.59 1.618 1.11 0.437 0.588 0.387 0.475 11 0.458 1.5812.134 2.608 4.105 1.747 1.736 0.985 1.663 11.5 0.822 0.978 2.596 1.692.76 1.784 2.968 3.548 3.922 11.7 0.411 3 2.416 3.004 3.865 3.716 2.4112.3 0.268 1.846 1.097 1.604 2.597 2.722 2.973 3.095 12.5 2.26 5.8614.385 4.566 4.8 8.507 6.208 5.664 12.7 0.636 1.145 0.79 1.642 1.502SubTotal 5.973 30.316 25.173 25.951 22.933 45.951 43.754 31.453 14.81613 1.261 0.521 1.421 1.556 1.99 2.785 2.495 13.1 1.124 1.769 2.61 1.3032.209 4.367 13.2 1.54 2.633 3.486 3.047 1.26 2.938 4.801 7.327 3.58413.4 1.331 1.122 2.196 1.805 3.72 3.161 3.088 13.6 1.383 0.783 1.3141.997 1.811 2.005 4.176 2.770 13.8 1.696 2.09 2.448 3.341 3.211 1.322.512 4.901 4.626 14 1.297 0.477 1.661 2.647 2.548 4.709 14.2 0.3790.726 0.78 0.963 0.942 2.779 1.74 0.762 SubTotal 3.615 11.845 11.35616.553 6.468 14.322 22.564 38.166 17.334 Total 9.588 42.161 36.52942.504 29.401 60.273 66.318 69.619 32.15 Total GC 11.98 47.12 54.17 77.5429.5 74.36 90.15 115.7 256.8 For 1 ml 38.6 152 175 250 1385 240 291 373828 conv. To umoles FA 0.385 1.52 1.75 2.5 13.85 2.4 2.91 3.73 8.28 Cal.AN 0.021 0.085 0.098 0.14 0.78 0.14 0.16 0.21 0.46

EXAMPLE 6 Genetic Engineering of an Alkaline Tolerant Microorganism forSurfactant Production

Sand Pack Columns:

Two strains of Pseudomonas were used as a host alkaline tolerantbacterium for the introduction of a test group of genes for heterologousexpression and biosurfactant production. The production of biosurfactantwas in media containing a simple carbon source and was not in contactwith petroleum oil. The production of biosurfactant was not linked toshort chain hydrocarbon metabolism.

Introduction of rhlAB Rhamnolipid Biosurfactant Production Genes intoAlkaliphilic Pseudomonas alcaliphila and Pseudomonas toyotomiensisStrains

The rhlAB operon from Pseudomonas aeruginosa PAO1 (ATCC BAA-47) encodesgenes for the production of a rhamnolipid biosurfactant. It has beendemonstrated that heterologous expression of the rhlA and rhlB genes,encoding a rhamnosyl transferase 1 enzyme, is sufficient for productionof mono-rhamnolipids with biosurfactant activity in E. coli (Ochsner etal 1994; Fang et al DOE report 2007) and P. putida (Wittgens 2011).

The rhlA and rhlB genes were introduced into, and heterologouslyexpressed in, alkaliphilic Pseudomonas species. The rhlA and rhlB geneswere amplified in a single PCR from Pseudomonas aeruginosa PAO1 genomicDNA using primers prGFF286 (containing a KpnI restriction site,ribosomal binding site, and the beginning of the rhlA ORF sequence) andprGFF287 (containing a XbaI restriction site, additional stop codon, andend of the rhlB ORF sequence). The resulting fragment was digested withKpnI and XbaI and was subsequently cloned into KpnI/XbaI-digestedplasmid pBBR1MCS, placing rhlAB gene expression under control of theLacZ promoter in pBBR1MCS. The resulting plasmid, pGFF88, was sequencedthroughout the insert and across the insertion junctions to verify thefidelity of the gene cloning. Plasmids pBBR1MCS and pGFF88 weretransformed into Pseudomonas alcaliphila (DSM 17744) and Pseudomonastoyotomiensis (JCM15604) by electroporation (modified from Sonnenscheinet al., 2011 J. Microbiological Methods). Briefly, cells were spreadplated onto two Mh-YCA plates (per liter: 25 g NaCl, 15 g agar, 5 gyeast extract, 5 g casamino Acids, 1 g sodium glutamate, 1 g NH4Cl, 1 gKH2PO4, 1 g KCl, 200 mg MgSO4.7H2O, 36 mg FeCl2.4H2O, 0.36 mg MnCl.4H2O,adjusted to pH7.0 with Na2CO3) and grown overnight at 30° C. Cells werescraped into a total volume of 4 mls B3 buffer (300 mM Glucose, 5 mMCaCl2, 25 mM HEPES, 5 mM MgCl2), spun 2 min at 20,000 g, and washedtwice with 1 ml ice cold B3 buffer. Cells were then resuspended in 200μl ice cold B3 buffer. An aliquot of 50 μl cells was mixed with 1 μlpBBR1MCS or pGFF88 DNA, transferred to an electroporation cuvette (2mm), and pulsed at 2 kV (10 kV/cm) and 900 μl SOC medium were added. Thesuspension was transferred to a culture tube, incubated 12 hours at 30°C., and spread plated onto Mh-YCA medium containing 400 μg/mlchloramphenicol. Plates were incubated at 30° C. Colonies were isolatedand checked by PCR, using primers flanking the pBBR1MCS multiple cloningsite and primers internal to the rhlAB sequence. Additionally, rDNAsequences were PCR amplified and sequenced, confirming that the isolateswere the appropriate Pseudomonas species.

Production of rhamnolipid biosurfactant was tested in engineered strainsusing the oil spreading assay (Fang et al DOE report 2007). Cells weregrown three days at 30° C. in YCA-10 medium (per liter: 25 g NaCl, 5 gyeast extract, 5 g casamino Acids, 5 g Na2CO3, 1 g sodium glutamate, 1 gNH4Cl, 1 g KH2PO4, 1 g KCl, 200 mg MgSO4.7H2O, 36 mg FeCl2.4H2O, 0.36 mgMnCl.4H2O, adjusted to pH10.0 with KOH) containing 5 μg/mlchloramphenicol. For oil spreading assays, 50 mls of distilled water wasdispensed into a 15 cm petri dish and 50 μl of petroleum oil was addedto the surface of the water. A 10 μl drop of medium or culturesuspension was added to the surface of the oil, and the diameter of theclear zone (if any) was measured after 2 minutes. Results are shownbelow in Table 3.

TABLE 3 Clear zone Sample Description diameter (mm) Medium Mh-YCA (pH7)<1 Medium YCA, pH10 <1 Surfactin (purified)- 13 100 μg/ml GFF255 P.alcaliphila + pBBR1MCS 1 GFF253 P. alcaliphila + pGFF88 29 GFF257 P.toyotomiensis + pBBR1MCS 2 GFF261 P. toyotomiensis + pGFF88 30

Production of rhamnolipid was tested in culture supernatants of six dayold cultures in the resulting P. alcahphila strain harboring the rhlABvector (GFF 253) using the orcinol assay (as described in Fang et al DOEreport 2007). Results were compared to assay standards made frompurified rhamnolipid (Sigma-Aldrich), which indicated that the strainproduced between 50 mg/L and 100 mg/L rhamnolipid.

Sand Pack Columns:

To test the ability of the rhamnolipid biosurfactant producingengineered strains, a six day old culture was tested for its ability toelute oil from a small sand pack column. One small sand pack column wasabout 50 ml in volume and was made by filling water containing glasscolumn with dry 30 mesh sand. The pore volume was determined bymeasuring the empty weight of the column, the dry weight of the sandneeded to fill the column and the final weight of the water filled sandcolumn. Two columns were prepared this way, with column #1 having a porevolume of 21.5 ml and the other column #2 having a pore volume of 22.8ml. Each column was filled with oil by pumping oil into the bottom ofthe vertical columns. The petroleum used in this experiment wasmoderately heavy (API 24) petroleum from Ecuador. The amount of oilpumped into the columns was determined by weight to be 15.4 g for column#1 and 15.0 g for column #2. At a density of 0.91 the oil filled 16.9 mlof the 21.5 ml pore volume in column #1 (79%) and 16.5 ml of the 22.8 mlpore volume in column #2 (72.4%).

The oil was then eluted off with water at a flow rate of about 6ml/minute. After about 30 pore volumes of water the weight of the oileluted was determined. The amount of oil eluted from column #1 was 10.57g or 68.6% of the oil that was applied. The amount of oil eluted fromcolumn #2 was 8.73 g or 58.2% of the oil that was applied. Next, a pH10.0 buffer was pumped into each column for another 30 pore volumes todetermine if more oil could be eluted with a higher pH buffer. Theadditional weight of the oil eluted by the 30 pore volumes was 0.38 gfrom column #1 and 0.23 g from column #2. Next a 50 ml aliquot of eachculture was pumped through each column at the same flow rate for twohours which was approximately 30 pore volumes of fluid flow. Therhamnolipid biosurfactant-producing P. alcaliphila strain, harboring therhlAB vector (GFF 253) was applied to column #1. The P. alcaliphilastrain harboring an empty vector without the rhlA and rhlB genes (GFF255) was applied to column #2. The amount of oil recovered frombiosurfactant producing strain was an additional 0.57 g and the amountproduced from the empty vector control (GFF 255) was 0.25 g. Anadditional two days of flow of each microbe containing solutions elutedanother 0.32 g from column #1 and an additional 0.61 g from column #2.At the end of the experiment, each column was extracted with a mixtureon hexane and toluene. The extracted mixture was dried with the flow ofwarm air (about 50° C.) for several days to remove the hexane andtoluene. The weight of each sample was used to determine the mass ofeach column. The total weight of solvent extracted oil was 2.65 g or17.2% of the original oil applied to column #1 and 3.31 g or 22.1% fromcolumn #2. The total oil recovered from column #1 was 11.84 g or 76.9%by aqueous elution and 14.49 g or 94.1% of the oil applied by totalextraction. The total oil recovered from column #2 was 9.82 g or 65.5%by aqueous elution and 13.13 g or 87.5% of the oil applied by totalextraction.

The amount of oil eluted immediately following the application ofsurfactant-producing microbes was greater than in the application on themicrobes with the empty vector, which did not exhibit the presence ofsurfactant in the oil spreading assay. However, during the subsequenttwo days of elution after the initial application of microbes, slightlymore oil was eluted with the empty vector control (GFF255 versusGFF253). The amount of oil eluted by the rhamnolipid was fairly smalland may have been limited by the low level production (50 to 100 mg/1)or by the fact that rhamnolipid has lower activity at a high pH and isbetter suited for a neutral or slightly acidic pH. To evaluate the pHeffect, a larger sand pack column with a volume of 250 ml was run at pH7.

The larger column was packed the same way as the smaller column and had422.5 g of sand with a water pore volume of 101.1 ml in column #1 and427.1 g of sand and a water pore volume of 100.8 ml in column #2. Eachcolumn was filled with oil by pumping from the bottom until oil startedto elute from the top. A total of 88 g of oil was pumped onto column #1and 83.5 g to column #2. The oil was left on the column for two daysbefore applying the media buffer solutions. For the lager columns theflow rate was reduced to 1.0 ml/minute and each 50 ml eluted for thefirst two pore volumes (200 ml) was collected. The amount of oil wasdetermined by removing the water from each collection tube and weighingthe remaining oil. The fluid used in both columns was the pH 7 mediathat the microbes were grown in. The next 500 ml, or 5 pore volumes,were collected as 100 ml samples and the weight of the oil determinedthe same way. At the end of pore volume number 7, each of the twodifferent bacterial cultures was applied to one of the two columns. Thecells producing rhamnolipid (GFF253) were applied to column #1 and theempty vector control cells (GFF255) were applied to column #2. The nexttwo pore volumes were collected as 50 ml samples, and the weight of oileluted was determined by removing the water as before. The next fourpore volumes were eluted in two collections of two pore volumes each.The amount of oil in each sample was determined and is listed in Table 4below. At the end of the aqueous elution experiment, the columns weredried with air flow and then extracted with 100 ml of toluene. Thetoluene was dried with the flow of warm air until all the volatilesolvent was removed. The final weight of the toluene extract after a fewdays' drying indicated the amount of unrecovered oil remaining on thecolumns after the bacterial extractions.

The results of the neutral pH column were similar to the smaller high pHcolumn. The bacteria producing the rhamnolipid eluted more oil in thefirst pore volume following the injection of cells. The control microbes(GFF255), containing an empty vector without genes coding for theproduction of rhamnolipid, appeared to have had a delayed affect on oilelution. This may have been due to the higher cell density of GFF255compared to the GFF253 strain (4.25 optical density at 600 nm vs. 2.48for the GFF253 strain).

The amount of oil actually eluted as a result of the rhamnolipid wassmall. While the gene was successfully transferred into this alkalinetolerant strain, as shown above, the amount of oil elution was small.This may be due to low expression levels or the ineffectiveness of thisparticular biosurfactant at high pH. However, this is a useful way tomeasure the benefit of various biosurfactants for oil recovery.

TABLE 4 Pore volumes Col. # 1 Total g of Col. #2 Total g of Col. #1 % ofoil Col. # 2 % of oil eluted oil recovered oil recovered recoveredrecovered 0.5 43.86 43.12 49.8 51.6 1.0 49.06 49 55.75 58.7 1.5 52.351.9 59.4 62.2 2 54.5 54.1 61.9 64.8 3 58.6 57.5 66.6 68.9 4 61.3 60.169.6 72 5 63.3 64.2 71.9 76.9 6 64.7 65.8 73.5 78.8 7 65.5 66.8 74.4 807.5 Add Cells 65.9 67.2 74.9 80.5 8 67 67.5 76.1 80.8 8.5 70 68.1 79.581.6 9 70.6 69.5 80.2 83.2 11 71 71.7 80.7 85.9 13 71.2 74 80.9 88.6Toluene Extract 12 9.9 13.6 11.8

EXAMPLE 7 The Introduction of Other Biosurfactant Production Pathwaysinto Other Alkaline Tolerant Microbes

Introduction of Rhamnolipid Biosurfactant Production Genes intoAlkalitolerant Bacillus halodurans

The rhlA and rhlB were introduced into Bacillus halodurans strain C-125(JCM9153), an alkalitolerant strain that grows under high pH conditionsof over pH11. The rhlA and rhlB genes were amplified in a single PCRfrom Pseudomonas aeruginosa PAO1 genomic DNA using primers prGFF296(containing the beginning of the rhlA ORF) and prGFF300 (containing aSacI restriction site, an additional stop codon, and the end of the rhlBORF sequence). A DNA fragment containing a SacI restriction site, thehag (sigmaD) promoter, and the beginning of the rhlA gene was generatedby PCR from B. halodurans genomic DNA using primers prGFF294 (containinga SalI restriction site and sequence upstream of the hag ORF) andprGFF295 (containing reverse complement sequence to the beginning of therhlA ORF followed by reverse complement sequence to hag promotersequence immediately upstream of the hag ORF). In a subsequent PCR, thetwo fragments were joined and amplified as a single fragment, usingprimers prGFF294 and prGFF300. The resulting fragment was digested withSalI and SacI and was subsequently cloned into SalI/SacI-digestedplasmid pNW33N. The resulting plasmid, pGFF94, was sequenced throughoutthe insert and across the insertion junctions to verify the fidelity ofthe gene cloning. Plasmids pNW33N and pGFF94 were transformed into B.halodurans JCM9153 and plated on succinate nutrient agar mediumcontaining 3 μg/ml chloramphenicol as described previously (Wallace2011). Colonies were isolated. PCR confirmed the presence of the plasmidin isolates, using primers flanking the pNW33N multiple cloning site andprimers internal to the rhlAB sequence.

Introduction of Surfactin Biosurfactant Production Genes intoAlkalitolerant Bacillus halodurans

In one example, a DNA segment containing the operon for surfactinproduction, including srfAA, srfAB, comS, srfAC, srfAD, ycxA, ycxB,ycxC, ycxD, and sfp from Bacillus subtilis strain ATCC21332, areintroduced into Bacillus halodurans strain C-125 (JCM9153), analkalitolerant strain that grows under high pH conditions of over pH 11.The DNA segment is amplified in a single PCR from Bacillus subtilisstrain ATCC21332 genomic DNA using primers prGFF314 (containing an SbfIrestriction site and sequence upstream of the srfAA ORF) and prGFF315(containing a SwaI blunt restriction site sequence downstream of sfp ORFsequence). The resulting fragment is digested with SbfI and SwaI andsubsequently cloned into SbfI/SmaI-digested plasmid pNW33N. Theresulting plasmid is introduced into Bacillus halodurans strain C-125(JCM9153) by established methods. Alternatively, a DNA segmentcontaining the same genes are introduced into Bacillus halodurans strainC-125 (JCM9153) under the regulation of a different promoter for optimalexpression in Bacillus halodurans. For example, the promoter upstream ofthe hag gene from Bacillus halodurans strain C-125 (JCM9153) isamplified by PCR using primers prGFF313 (containing a hindIIIrestriction site followed by sequence matched to the 5 prime end of thepromoter) and prGFF312 (containing a SbfI restriction site followed byreverse complement sequence matched to the hag promoter immediatelyupstream of the ribosomal binding site). The resulting fragment is cutand ligated into pNW33N at the HindIII and SbfI sites. A DNA segmentcontaining the operon for surfactin production, including srfAA, srfAB,comS, srfAC, srfAD, ycxA, ycxB, ycxC, ycxD, and sfp from Bacillussubtilis strain ATCC21332, is PCR amplified using prGFF311 (containingan SbfI restriction site, a ribosomal binding site, and sequence matchedto the beginning of the srfAA ORF) and prGFF315 (containing a SwaI bluntrestriction site sequence downstream of sfp ORF sequence). The resultingsegment is cut with SbfI and SwaI and cloned into the SbfI and SmaIsites in the pNW33N vector containing the hag promoter, placing thesurfactin operon under the regulation of the hag promoter. The resultingplasmid is introduced into Bacillus halodurans strain C-125 (JCM9153) byestablished methods.

TABLE 5 Primer name Sequence Notes prGFF286 CCTTGGAggtaccAGGAGGTTTTTATTaforward oligo for RhIAB amplification for pBBR1MCS tgcggcgcgaaagtctgttgg(SEQ ID NO: vectors (Kpnl site plus RBS plus other plus 11) beginning ofRhIAB) prGFF287 aaccaaggTCTAGAtcaTTAtcaggacgca reverse oligo for RhIABamplification for pBBR1MCS gccttcagccatcg (SEQ ID NO: 12) vectors (Xbalsite plus stop codon plus end of RhIAB) prGFF294ccttggaGTCGACctcgcgttacgctctttctgt extra sequence plus Sall plus hagpromoter forward (SEQ ID NO: 13) prGFF295ggcccttgcaaaccgataccaacagactttcgc Beginning of RhIAB (rc) plus hagpromoter sequence gccgcatTAAAATTTCCTCCTTGAAATG immediately before start(rc) TGTT (SEQ ID NO: 14) prGFF296 ATGCGGCGCGAAAGTCTGTTGGTATC start ofRhIAB (SEQ ID NO: 15) prGFF300 CGAgagctcAAGTAACGGTTGAGCGA Sacl site plussequence after hag (rc) plus AAAATAAAAAAGACCTGGAATAAAT extra stop plusend of RhIAB (rc) CCAGATCTTTAAATGGAAGCAAAttaT CAGGACGCAGCCTTCAGCCATC(SEQ ID NO: 16) prGFF311 AAAGACAAcctgcaggCGGAAAACACA Random sequenceplus Sbfl restriction site plus 30nt TTTCAAGGAGGAAATTTTAatggaaataimmediately upstream of hag c-125 ORF plus firstactttttaccctttaacggatgcacaa (SEQ 36nt in srfAA ORF ID NO: 17) prGFF312AACGTTTAcctgcaggCTTGTGGTTGGT Random sequence plus Sbfl site plussequence GTAAACTTAATCC (SEQ ID NO: 18) upstream of c-125 hag shinedalgarno site (rc) prGFF313 ccttggaAAGCTTctcgcgttacgctctttctgt Randomsequence plus HinDIII plus hag promoter (SEQ ID NO: 19) forward prGFF314aaagacaaCCTGCAGGagttacttggagga Forward primer for amplification of srfAoperon; tactgtgaggagagacaatcag (SEQ ID random sequence plus Sbflrestriction site plus NO: 20) sequence upstream of srfA1 ORF prGFF315cgatccatATTTAAATaaaattggggccctcc Reverse primer for amplification ofsrfA operon; tgaatatggtgctcattggtg (SEQ ID NO: random sequence plus Swalrestriction site plus 21) sequence downstream of sfp; note that sequencediffers between genbank sequences at prGFF315

The invention claimed is:
 1. A microorganism of the domain Archaea orbacteria, that is engineered to have at least one of the followingproperties: (i) being an obligatory alkaliphile, halo-alkaliphile oralkaline tolerant, (ii) being deficient in its ability to degrade shortchain hydrocarbons of 12 carbons or less, and (iii) having the abilityto produce surfactants, wherein the Archaea is of the genus Haloferax,and wherein the bacteria is selected from the group of genera consistingof Pseudomonas and Bacillus.
 2. The microorganism of claim 1 whereinsaid microorganism is engineered to have properties (i) and (ii).
 3. Themicroorganism of claim 1, that is of the domain Archaea.
 4. Themicroorganism of claim 1, that is a bacterium.
 5. The microorganism ofclaim 1, that is able to grow in alkalinity of pH 9.0 or higher.
 6. Themicroorganism of claim 1, that is able to grow in alkalinity of pH 10.0or higher.
 7. The microorganism of claim 1, that has the ability toutilize hydrocarbon chains of greater than 12 carbons.
 8. Themicroorganism of claim 1, that has the ability to utilize modifiedhydrocarbons containing sulfur.
 9. The microorganism of claim 1, thathas the ability to utilize modified hydrocarbons containing nitrogen.10. The microorganism of claim 1 wherein said microorganism isengineered to have properties (ii) and (iii).
 11. The microorganism ofclaim 1 wherein said microorganism is engineered to have properties (i),(ii), and (iii).
 12. The microorganism of claim 1, wherein the Archaeais Haloferax volcanii.
 13. The microorganism of claim 1, wherein thebacteria is Pseudomonas toyotomiensis.
 14. The microorganism of claim 1,wherein the bacteria is Pseudomonas alcaliphila.
 15. The microorganismof claim 1, wherein the bacteria is Bacillus halodurans.
 16. Aconsortium comprising one or more types of microorganisms according toany one of claims 1, 2, and 5 to
 9. 17. An oil reservoir comprising aconsortium of claim 16.